Magnesium oxalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro test (Ames test and in vitro gene mutation study in mammalian cells) show negative results with and without metabolic activation on oxalic acid. Considering the read across approach used for assessment, these results are considered reliable for assessing the mutagenic properties of magnesium oxalate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common magnesium cation: Mg2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of magnesium oxalate takes place immediately, resulting in formation of magnesium cations (Mg2+) and oxalate anions. Thus, any ingestion or absorption of magnesium oxalate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Magnesium is an abundant mineral naturally present in the body. It is a cofactor in more than 300 enzyme systems that regulate diverse biochemical reactions in the body, including protein synthesis, muscle and nerve function, blood glucose control, and blood pressure regulation (IOM 1997, Rude 2010, Rude 2012). Magnesium is required for the normal functioning of several biochemical and physiological reactions and pay an essential role in the human body (Rude 2012). An adult body contains approximately 25 g magnesium, with 50% to 60% present in the bones and most of the rest in soft tissues (Volpe 2012). Human Recommended Dietary Allowances (RDA) for magnesium is up to 420mg per day (IOM 1997). For the same reasons (involvement in biochemical and physiological functions), magnesium is also naturally present in various organisms of the environment such as fish, crustacea or vegetables. Besides they are identified as food sources of magnesium (US 2012). Consequently, it can be concluded that magnesium is of low (eco)toxicological relevance when ingested and taken up systemically. Thus, any possible toxicological or ecotoxicological effect triggered by magnesium oxalate exposure can be attributed to oxalate anion.
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (magnesium oxalate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Mg2+ or oxalate ion).

Tests on the mutagenic potential of magnesium compounds in bacteria are considered dispensable for principal considerations, since inorganic metal compounds are frequently negative in this assay due to limited capacity for uptake of metal ions (Guidance on information requirements and chemical safety assessment, Chapter R.7a, p. 565; HERAG facts sheet mutagenicity, Chapter 2.1).
Additionally, as discussed below the magnesium ion (Mg2+) is not the part of interest in the read-across purpose and is not envisaged. In the same manner than the other endpoints, this endpoint will be based on a read across based on the oxalate ion.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.

3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration dossier on strontium peroxide) confirms the analogue approach performed (same results on analogues).

4. DATA MATRIX
A data matrix is presented in the detailed report attached.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: S. typhimurium TA92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: S. typhimurium TA94

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

The following table is extracted from the table in the publication (only the part concerning oxalic acid is presented):

Additive	CAS no.	Purity (%)	Max dose (mg/plate)	Solvent	Result
Oxalic acid	144-62-7	99.7	10.0	Phosphate buffer	-

Conclusions:

Oxalic acid does not have mutagenic properties in the Ames test, under the current test conditions. According to the read across approach presented, magnesium oxalate have to be considered to not have mutagenic properties in the Ames test.

Executive summary:

200 food additives used in Japan, including oxalic acid, were tested for mutagenic properties in the Ames test. The strains of Salmonella typhimurium used are the following: TA 92, TA1535, TA100, TA1537, TA94, and TA98, with and without metabolic activation (S-9 prepared from rat liver). Negative results are showed with oxalic acid in all strains, with and without activation. It can be concluded that oxalic acid does not have mutagenic properties in the Ames test, under the current test conditions.

This resuslt is considered relevant in a read across approach and it is used for assessing the potential mutagenic property of magnesium oxalate. Following the read across approach, magnesium oxalate have to be considered to not have mutagenic properties in the Ames test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

July, 19 2016 to January 31 2017

Reliability:

1 (reliable without restriction)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common magnesium cation: Mg2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of magnesium oxalate takes place immediately, resulting in formation of magnesium cations (Mg2+) and oxalate anions. Thus, any ingestion or absorption of magnesium oxalate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Magnesium is an abundant mineral naturally present in the body. It is a cofactor in more than 300 enzyme systems that regulate diverse biochemical reactions in the body, including protein synthesis, muscle and nerve function, blood glucose control, and blood pressure regulation (IOM 1997, Rude 2010, Rude 2012). Magnesium is required for the normal functioning of several biochemical and physiological reactions and pay an essential role in the human body (Rude 2012). An adult body contains approximately 25 g magnesium, with 50% to 60% present in the bones and most of the rest in soft tissues (Volpe 2012). Human Recommended Dietary Allowances (RDA) for magnesium is up to 420mg per day (IOM 1997). For the same reasons (involvement in biochemical and physiological functions), magnesium is also naturally present in various organisms of the environment such as fish, crustacea or vegetables. Besides they are identified as food sources of magnesium (US 2012). Consequently, it can be concluded that magnesium is of low (eco)toxicological relevance when ingested and taken up systemically. Thus, any possible toxicological or ecotoxicological effect triggered by magnesium oxalate exposure can be attributed to oxalate anion.
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (magnesium oxalate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Mg2+ or oxalate ion).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.

3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration dossier on strontium peroxide) confirms the analogue approach performed (same results on analogues).

4. DATA MATRIX
A data matrix is presented in the detailed report attached.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

valid

Remarks:

DMEM Medium

Positive controls validity:

valid

Remarks:

-S9 Ethyl methanesulfonate, +S9 7, 12 Dimethyl benz(a)anthracene

Conclusions:

Conclusion
During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, OXALIC ACID is considered “non-mutagenic” in this HPRT assay, and in a read across approach, magnesium oxalate is also considered as “non-mutagenic”.

Executive summary:

This study was conducted to investigate the potential of OXALIC ACID to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The methods followed were as per OECD guideline No. 476, adopted on 28th July 2015.

The assay was performed in a independent experiment, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 4 hours with and 24 hours with out metabolic activation.

500 µg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item.

The following concentrations were selected for both Phase - I and Phase - II based on cytotoxicity results.

500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL both in the presence and absence of metabolic activation

No relevant cytotoxic effect as indicated by the relative survival (RS) is cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count and as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). The RS for the test item in the other tested concentrations were found to be more than 50% and hence the same doses were selected for the main experiment (Phase-I and Phase-II).

PHASE-I

In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 7.5, 8.7, 8.8, 12.9, 17.5, 23.8 and 152.1/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 4.3, 10.3, 11.7, 15.6, 24.7, 28.1 and 1181.2 per 106 cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 5.8, 9.5, 14.5, 15.7, 17.2, 26.9 and 169.9/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 9.3, 12.8, 17.6, 17.8, 24.7, 28.7 and 1387.6 per 106 cells in presence of metabolic activation.

PHASE-II

In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.6, 6.9, 7.8, 9.9, 15.7, 20.9 and 170.6/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 5.2, 6.7, 10.2, 15.1, 19.0, 23.2 and 1022/106 cells in presence of metabolic activation.

In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.0, 7.5, 10.0, 13.3, 17.4, 23.7 and 155.2/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 7.4, 10.7, 12.5, 17.3, 22.1, 25.2 and 1211.6 per 106 cells respectively in presence of metabolic activation.

In both the cultures, there was no distinct increase in the mutant frequency of OXALIC ACID when compared to respective vehicle control and the induction factor not exceeds more than three times the corresponding vehicle controls. No significant and reproducible dose dependent increase in mutant colony numbers was observed in either the Phase I or Phase II of the experiment.

The positive controls used, EMS in the absence of metabolic activation and DMBA  in the presence of metabolic activation, revealed significant increase in mutant colonies and induction factor is more than three times of vehicle control indicating that the test system were sensitive and the results are valid

Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; T4: Test concentration 4; PC: Positive Control, EMS (ethyl methanesulfonate), DMBA (Dimethyl benz(a)anthracene)

The result of this study is considered reliable in a read across approach for magnesium oxalate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No mutagenic effect was observed on the test item. In a read across approach, these results are considered relevant for assessing the classification of magnesium oxalate and it is thus considered that there is no need for classification.