Magnesium, EDTA cobalt copper iron manganese zinc complexes

Endpoint summary

Additional information

1.   Hypothesis for the analogue approach

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.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across). In this case the related substances mentioned below are ingredients ofMagnesium, EDTA cobalt copper iron manganese zinc complexes(CAS 234446-82-3).

 

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

 

	Target Substance	Source Substance No 2	Source Substance No 3	Source Substance No 4
Name	Magnesium, EDTA cobalt copper iron manganese zinc complexes	EDTA-MnNA2	EDTA-FeNa	EDTA-CuNa2
CAS	234446-82-3	15375-84-5	15708-41-5	14025-15-1
% in CAS 234446-82-3	n.a.	30	25	9
In vitro gene mutation in bacteria	Negative	n.r.	n.r.	n.r.
In vitro gene cytogenicity in mammalian cells	--	Negative	MN: Aneugenic but no clastogenic   MLA (TK): Negative	MN: Aneugenic but no clastogenic

 

n.a.: not applicable; n.r.: not required, because study with target substance is available; lack of data for a given endpoint is indicated by “--“.

 

2.   Analogue approach justification

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information of the source substances is used to predict the respective endpoints forMagnesium, EDTA cobalt copper iron manganese zinc complexes(CAS 234446-82-3).

 

Since only an in vitro gene mutation study in bacteria is available forMagnesium, EDTA cobalt copper iron manganese zinc complexes(CAS 234446-82-3),a read-across for in vitro cytogenicity and gene mutation in mammalian cells was performed from the structurally related analogue substances (which are components of CAS 234446-82-3) EDTA-MnNa2 (CAS15375-84-5),EDTA-FeNa (CAS 15708-41-5) and EDTA-CuNa2 (CAS 14025-15-1) was performedin accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5.

 

3.   Results

a.   Target Substance: KEY STUDY

EDTA cobalt copper iron manganese zinc complexes(CAS 234446-82-3)

The test substance Magnesium, EDTA cobalt copper iron manganese zinc complexes was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in the AMES test according to OECD guideline No 471 with and without S9 -mix under GLP conditions (BASF 2013).

The used strains were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 with dose ranges from 33 µg - 6000 µg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance Magnesium, EDTA cobalt copper iron manganese zinc complexes is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

b.   Source substances

-Source Substance No1: EDTA-MnNa2 (CAS 15375-84-5)

The test substance EDTA-MnNa2 was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix), according to Draft OECD Guideline 487 (deVogel, 2010). Two separate in vitro micronucleus tests were conducted for which blood was obtained from two different donors. In the first test, in the presence and absence of S9 -mix, the treatment/recovery time was 4/20 hours (pulse treatment method). In the second test, concentration spacing was modified and the treatment/recovery times were 20/28 hours (continuous treatment) in the absence of S9-mix. Dose levels, ranging from 7.8 to 3891 µg/ml, were tested. In all instances, the maximum final concentration in the culture medium was 10 mmol/l. In the first test, in the presence and absence of S9-mix, the highest dose level (3891 µg/ml) was cytotoxic to the cells, but the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

In the second test, the two highest dose levels (3000 and 3891 pg/ml) were cytotoxic to the cells, and again the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

In vitro micronucleus tests showed that, under the conditions used in the study, the test substance EDTA-MnNa2 was neither clastogenic nor aneugenic to cultured human lymphocytes.

 

-      Source Substance No2: EDTA-FeNa (CAS 15708-41-5)

a)    Micronucleus Test

The test substance EDTA-FeNa was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) , according to Draft OECD Guideline 487 (deVogel, 2010). The micronucleus study consisted of two separate tests for which blood was obtained from two different donors. In the second test, size-classified micronucleus counting was additionally performed on the slides of three dose levels of the test substance and the positive controls Mitomycin C and Vinblastin sulphate, to discriminate aneugens from clastogens.

In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment time was 4 hours (pulse treatment) and the recovery time 20 hours. In the second test, in which metabolic activation was absent and concentration spacing was modified, the treatment time was 20 hours (continuous treatment) and the recovery time 28 hours. Dose levels of the test substance ranging from 7.8 to 4211μg/ml were tested in the culture medium. In all instances, the maximum final concentration in the culture medium was 10 mmol/l.

In the first test, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the dose levels analysed.

In the second test,  the test substance induced a statistically significant increase  in the number of binucleated cells containing micronuclei at the three highest concentrations (500, 1500, 2500 µg/ml), but not at the lowest dose tested (125μg/ml).

Size-classified micronucleus counting revealed that the test substance EDTA-FeNa clearly increased the proportion of large micronuclei at all dose levels analysed.

Based on the results obtained in two in vitro micronucleus tests, the test substance EDTA-FeNa induced a statistically significant increase in the number of binucleated cells containing micronuclei in the second test (continuous treatment) under the conditions used in this study. Based on the results of the size-classified micronucleus counting in the second test (continuous treatment), EDTA-FeNa clearly increased the proportion of large micronuclei at three dose levels which is considered to be an indication for aneugenic effects, under the conditions used in this study.

b)   Mouse lymphoma test

The mutagenic activity of EDTA-FeNa and EDTA-Na2 was tested in a mammalian gene mutation assay with L5178Y mouse lymphoma cells. EDTA-FeNa was positive in the mouse lymphoma mutagenicity study, but only at test concentrations that caused significant cytotoxicity. OECD guideline No 476 cites: "Care should be taken to avoid conditions which would lead to results not reflecting intrinsic mutagenicity. Positive results which do not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity." Therefore the results are ambiguous. EDTA-Na2 is much less cytotoxic and is also negative in this assay.

 

-      Source Substance No3: EDTA-CuNa2 (CAS 14025-15-1)

The test substance EDTA-CuNa₂ was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) according to the OECD guideline 487 (Usta, 2013). Two independentin vitromicronucleus tests were conducted for which blood was obtained from two different donors. Culture medium was used as solvent for the test substance. Dose levels ranging from 7.8 to 3977 µg/ml (3977 µg/ml = 10 mM) were tested in duplicates as final concentrations in the culture medium. In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment).

In the second test, in the continuous treatment group without metabolic activation, the treatment/recovery time was 20/28 hours.

In the pulse treatment group with metabolic activation the test substance did not show a significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed.

In the continuous treatment group without metabolic activation, the test substance induced a dose dependent statistically significant increase in the number of binucleated cells containing micronuclei.

Size-classified micronucleus counting revealed, that the proportion of the large micronuclei and small micronuclei found at the three test substance dose levels (250, 125 and 62.5µg/ml) analysed was not statistically different from the response of Vinblastine Sulphate at acceptable toxicity levels. The observed similar proportions of large and small micronuclei compared to the aneugen Vinblastine Sulphate, is considered to be an indication for an aneugenic effect, under the conditions used in this study.

 

 

Key study assignment:

There is one in vitro gene mutation in bacteria available for the target substance, which is well documented, reliable and was conducted in general accordance with the OECD principles of GLP. Therefore this study (BASF, 2013) was assigned as key study.

 

Conclusion

An in vitro gene mutation in bacteriatest with the target substanceEDTA cobalt copper iron manganese zinc complexes(CAS 234446-82-3) was performed, providing negative results (BASF, 2013).

Anin vitro gene mutation in mammalian cells with the source substanceEDTA-MnNa2 (CAS 15375-84-5) was negative.

 

EDTA-FeNa was also negative in the Ames test (Dunkel et al, 1999), in the WP2 Mutoxitest (Martinez et al, 2000) and in the in vitro micronucleus test using a treatment period of 4 h (with and without S9 -mix) (see above). In a mouse lymphoma assay (see above) with EDTA-FeNa, increases in mutants were observed but only at high cytotoxic concentrations. Not taking the cytotoxic concentrations into account, no increase in mutant incidences above control values were seen. The absence of mutagenicity in the Ames test for the same compound suggests that mouse lymphoma cells may be particularly sensitive to incorporation of excessive quantities of iron salts in the tissue culture growth medium. In the in vitro micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix), EDTA-FeNa was positive at levels >= 500 µg/mL, inducing aneugenic but no clastogenic effects.

Two Chinese in vivo genotoxicity studies (Sichuan Provincial Sanitary and Anti-epidemic Station, 1993), although with major flaws such as lack of details, no GLP, and no official translation, however, did not show genotoxicity in vivo.

 

EDTA-CuNa2 was negative in the Ames test (BASF, 1992) and in the in vitro micronucleus test using a treatment period of 4 h (with and without S9 -mix) (see above). In the in vitro micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix) (see above), EDTA-CuNa2 was positive at levels >= 62.5 µg/mL, inducing aneugenic but no clastogenic effects.

Although no in vivo genotoxicity studies have been carried out with EDTA-CuNa2, several in vivo genotoxicity studies are available for other EDTA-compounds such as EDTA-Na2H2. No genotoxic activity was observed.

 

The long treatment period in the continuous treatment experiments with EDTA-FeNa and EDTA-CuNa2 together with the high concentrations of chelants may have resulted in exchange and substantial binding of essential elements such as zinc. Heimbach et al (2000) concluded that the lack of effects by the Zn-EDTA salt in contrast to effects induced by Ca-, Na- and Mn-salts of EDTA, provided evidence that zinc is required for the initiation or continuation of DNA synthesis and maintaining cell function. As such, the significance of mutations produced by EDTA-CuNa2 and also EDTA-FeNa at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms.

 

Therefore, the overall findings indicate that EDTA-CuNa2 and EDTA FeNa lack significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function.

 

Altogether, it is concluded, that the target substance EDTA cobalt copper iron manganese zinc complexes (CAS 234446-82-3) is not mutagenic.

 

References:

BASF (1992), Report on the study of Trilon B-CU 1550 in the Ames test (Salmonella/Mammalian-Microsome Mutagenicity Test - Standard Plate Test and Preincubation Test), RepNo. 40M0401/914239

BASF (2013), Magnesium, EDTA cobalt copper iron manganese zinc complexes, Reverse mutation assay, RepNo. 40M0361/13M132

Dunkel VC, San RHC, Seifried HE, Whittaker P (1999) Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells. Environ Mol Mutagen 33, 28-41

 

Heimbach J, Rieth S, Mohamedshah F, Slesinski R, Samuel-Fernando P, Sheenan T, Dickmann R, Borzelleca J (2000) Safety Assessment of Iron EDTA [Sodium Iron {Fe+3} Ethylenediaminetetraacetic Acid]: Summary of Toxicological, Fortification and Exposure

Data. Food Chem Toxicol 38: 99-111

 

Martinez A, Urios A, Blanco M (2000), Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyR+ parent WP2 uvrA/pKM101: detection of 31 oxidative mutagens, Mutation Res 467, 41-53

Sichuan Provincial Sanitary and Anti-epidemic Sstation (1993), Tests and results in toxicological evaluation on safety of NaFeEDTA, Translation of an unpublished report provided by Junshi Chen, Beijing, Chinese Center for Disease Control and Prevention

Usta B (2013a) In vitro micronucleus test with EDTA-CuNa2 in cultured human lymphocytes. TNO Triskelion, Zeist, the Netherlands, Report no. V20217/03

 

Vogel de N, Usta B (2010) In vitro micronucleus test with EDTA-FeNa in cultured human lymphocytes, TNO Quality of Life, Zeist, The Netherlands, Report no. V8817/07

 

 

Short description of key information:
The overall findings indicate that EDTA-CuNa2 and EDTA FeNa lack significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function.

Altogether, it is concluded, that the target substance EDTA cobalt copper iron manganese zinc complexes (CAS 234446-82-3) is not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the information received for the target substance and structurally similar source substances, no classification is derived according to CLP-Regulation (EC) 1272/2008 or Directive 67/548/EEC,

GHS: no classification

DSD: no classification