Reaction products of sodium glucoheptonate with copper sulfate and ammonium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity in bacterial cells can be expected for Copper glucoheptonate since all in vitro tests (key and supporting studies) in bacterial cells conducted with the structural analogues gluconates and inorganic copper compounds were negative. Additionally, in vivo chromosome aberrations studies conducted with glucono-delta-lactone and sodium gluconate were negative and indicated that no genetic toxicity can be attributed to gluconate and glucoheptonate ions.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented publication which meets basic scientific principles.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mutagenicity of copper gluconate, a dietary supplement, was examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemical was tested with and without S9 mix.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

0. 0.01, 0.05, 0.1, 0.5 and 1.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffer (pH 7.4).

Untreated negative controls:

yes

Remarks:

Phosphate buffer (pH 7.4)

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Remarks:

in TA97

Positive control substance:

9-aminoacridine

other: 2-AA

Remarks:

-S9 mix: 9-AA (30 µg); +S9 mix: 2-AA (5 µg)

Untreated negative controls:

yes

Remarks:

Phosphate buffer (pH 7.4)

Negative solvent / vehicle controls:

yes

Remarks:

negative control

True negative controls:

no

Positive controls:

yes

Remarks:

in TA102

Positive control substance:

mitomycin C

other: 2-AA

Remarks:

-S9 mix: MMC (0.5 µg); +S9 mix: 2-AA (5 µg)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min

Statistics:

Statistically significant defference by Kruskal-Wallis test ( p <0.05) and dose-related increasing by regression analysis (p < 0.01).

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Results of Mutation Test with copper (II) gluconate

Dose (mg/plate)	No of Revertants /plate
	TA97		TA102
	-S9	+S9	-S9	+S9
1	105	106	82	205
0.5	108	127	114	317
0.1	120	121	220	396
0.05	116	133	266	394
0.01	135	139	253	389
0	124	173	238	432
Positive control	179	2523	4105	1668
Solvent: DW (not specified)

Conclusions:

Copper gluconate was negative in two tester strains Salmonella typhimurium TA97 and TA102.

Executive summary:

Mutagenicity of 28 food additives including 7 dietary supplements, 7 free flowing agents, 5 antioxidants, 3 thickening agents, 3 food colors, 2 color fixatives, and an anticaking agent were examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. Copper gluconate was negative in two tester strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

D-Glucono-1,5 -Lactone, Sodium gluconate

The frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administartion and 4-d repeat administration. Thus, D-Glucono-1,5 -Lactone and Sodium gluconate are not genotoxic. This is relevant for copper glucoheptonate, because gluconate moiety is very similar to glucoheptonate moiety. Therefore, no genotoxicity can be attributed to the glucoheptonate moiety of copper glucoheptonate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented peer-reviewed report.

Qualifier:

no guideline available

GLP compliance:

no

Remarks:

the study was conducted prior to adoption of guidelines (in 1974).

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

C57BL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 or 13 weeks

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline;
- Amount of vehicle (if gavage or dermal): 1 mL/mouse

Duration of treatment / exposure:

single dose and 4 days

Frequency of treatment:

not specified

Post exposure period:

The animals were sacrified at 24 hours (single dose) and 27 hours after last administration (4-days repeated dose).

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

4 000 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

8 000 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

4-day repeated dose administration

Dose / conc.:

4 000 mg/kg bw/day

Remarks:

4-day repeated dose administration

No. of animals per sex per dose:

Single dose administration: 3 (vehicle control and test groups); 2 (positive control);
4-day repeated dose administration: 2 (vehicle control); 3 (test group 1: 4 g/kg); 2 (test group 2: 2 g/kg); 2 (positive control).

Control animals:

yes, concurrent vehicle

Positive control(s):

MMC (mitomycin C) dissolved with 0.9% physiological saline solution and administered intraperitoneally at a dose of 0.5 mL/mouse (= 5 mg/kg bw).

Tissues and cell types examined:

At least 200 metaphase cells per mouse were examined for the presence or absence of chromosomal aberrations (gaps, breaks, translocation, fragments, ring chromosomes and minutes chromosomes).

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: After receiving the single dose and the repeated dose test substance, the animals were sacrified at 24 hours (single dose) and 27 hours after last administration (4-days repeated dose). 0.3 mL of 500 μg/mL colchicine was intraperitoneally injected to each mouse at one hour before sacrifice so that the metaphase cells could be observed.

DETAILS OF SLIDE PREPARATION: After the bone marrow cells were washed, treated and fixed with a fixing solution (1:3 acetic acid:ethanol solution), the cells were suspended and dripped on a slide glass and stained with Giemsa solution and examined.

Sex:

male

Genotoxicity:

negative

Remarks:

in both experiments: single administration and 4-d repeated dose administration.

Toxicity:

yes

Remarks:

At 8 g/kg, all mice died (single dose administration experiment)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Single dose administration:

At 8 g/kg, all mice died.

MMC induced chromosomal aberrations in at least 20% of bone marrow cells.

GDL induced chromosomal aberrations in the cells at a frequency of about 0.5% comparable to the control.

4-day repeated dose administration:

MMC induced chromosomal aberrations at about 30% cells.

The frequency of cells with chromosomal aberrations was 1 % or less in the test groups which is comparable to the control group. Induction of chromosomal aberration by GDL was not detected after in vivo single and repeated dose treatment.

Conclusions:

The frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administartion and 4-d repeat administration. Thus, D-Glucono-1,5-Lactone is not clastogenic.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented peer-reviewed reports.

Qualifier:

no guideline available

GLP compliance:

no

Remarks:

the study was conducted prior to adoption of guidelines (in 1974).

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

C57BL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 or 13 weeks

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline;
- Amount of vehicle (if gavage or dermal): 1 mL/mouse

Duration of treatment / exposure:

single dose and 4 days

Frequency of treatment:

not specified

Post exposure period:

The animals were sacrified at 24 hours (single dose) and 27 hours after last administration (4-days repeated dose).

Dose / conc.:

2 500 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

5 000 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

10 000 mg/kg bw/day

Remarks:

single dose administration

Dose / conc.:

1 250 mg/kg bw/day

Remarks:

4-day repeated dose administration

Dose / conc.:

2 500 mg/kg bw/day

Remarks:

4-day repeated dose administration

No. of animals per sex per dose:

Single dose administration: 3 (vehicle control and test groups); 2 (positive control);
4-day repeated dose administration: 2 (vehicle control); 3 (test group 1: 2.5 g/kg); 2 (test group 2: 1.25 g/kg); 2 (positive control).

Control animals:

yes, concurrent vehicle

Positive control(s):

MMC (mitomycin C) dissolved with 0.9% physiological saline solution and administered intraperitoneally at a dose of 0.5 mL/mouse (= 5mg/kg bw).

Tissues and cell types examined:

At least 200 metaphase cells per mouse were examined for the presence or absence of chromosomal aberrations (gaps, breaks, translocation, fragments, ring chromosomes and minutes chromosomes).

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: After receiving the single dose and the repeated dose test substance, the animals were sacrified at 24 hours (single dose) and 27 hours after last administration (4-days repeated dose). 0.3 mL of 500 μg/mL colchicine was intraperitoneally injected to each mouse at one hour before sacrifice so that the metaphase cells could be observed.

DETAILS OF SLIDE PREPARATION: After the bone marrow cells were washed, treated and fixed with a fixing solution (1:3 acetic acid:ethanol solution), the cells were suspended and dripped on a slide glass and stained with Giemsa solution and examined.

Sex:

male

Genotoxicity:

negative

Remarks:

in both experiments: single dose administration and 4-d repeated dose administration.

Toxicity:

yes

Remarks:

At 10 and 5 g/kg, all mice died (single dose administration); at 1.25 and 2.5 g/kg, one mouse died in each group (4-day repeated dose administration).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Single dose administration:

At 10 and 5 g/kg, all mice died.

At 2.5 g/kg, observation could be made only on 2 animals (preparation of the chromosome specimen failed).

MMC induced chromosomal aberrations in at least 20% of bone marrow cells. Sodium gluconate induced chromosomal aberrations in the cells at a frequency of about 0.5% is comparable to the control. (1 gap and 1 minute chromosome for 283 cells).

4-day repeated dose administration:

At 1.25 and 2.5 g/kg, one mouse died in each group.

MMC induced chromosomal aberrations at about 30% cells. The frequency of cells with chromosomal aberrations was 0.5% in the test groups which is comparable to the control group.

Conclusions:

Induction of chromosomal aberration by sodium gluconate was not detected after in vivo single and repeated dose treatment. Thus, sodium gluconate is not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no genetic toxicity in vitro studies available for Copper glucoheptonate. Copper glucoheptonate is a chelate compound consisting of two molecules of sugar-like glucoheptonic acid and two copper ions. Data on structurally similar gluconic acid, its derivatives and organic and inorganic copper compounds have been taken into account to assess mutagenicity potential of Copper glucoheptonate in bacterial cells.

An in vitro genetic toxicity study with Copper gluconate

Mutagenicity of 28 food additives including 7 dietary supplements, 7 free flowing agents, 5 antioxidants, 3 thickening agents, 3 food colors, 2 color fixatives, and an anticaking agent were examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102 (Fujita et al., 1988). The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. The concentrations tested were: 0. 0.01, 0.05, 0.1, 0.5 and 1.0 mg/plate. Copper gluconate was negative in two tester strains.

In vitro genetic toxicity studies with gluconates and their derivatives

Sodium gluconate, glucono-delta-lactone and calcium gluconate were tested by plate incorporation method and in suspension on Saccharomyces cerevisiae and Salmonella typhimurium with and without metabolic activation (SIDS, 2004). OECD Guideline 471 was deviated for the number of strains tested and the choice of positive controls. The substances were tested on Saccharomyces cerevisiae (strain D4) and Salmonella typhimurium (3 strains: TA 1535, TA 1537, TA 1538) with and without metabolic activation. Only 3 concentrations were tested, while the OECD guideline recommends at least 5 concentrations. None of the test substances showed mutagenicity on the strains tested.

The genotoxicity of a number of sugar-like substances has been evaluated in in vitro and in vivo studies. The results of these studies are overwhelmingly negative (CIR, 2014).

In vivo genetic toxicity studies with glucono-delta-lactone and sodium gluconate.

Since glucono-delta-lactone, sodium and calcium gluconates were tested only in three strains of Salmonella typhimurium with three concentrations, the results of in vivo studies conducted with glucono-delta-lactone and sodium gluconate (SIDS, 2004) can be taken into account to assess genetic toxicity potential of glucoheptonate ion.

Glucono-delta-lactone and sodium gluconate did not induce chromosomal aberrations in mice. The frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administartion and 4-d repeat administration.

In vitro genetic toxicity studies with inorganic copper compounds

Copper compounds were not mutagenic in most studies in bacteria and yeasts (SCOEL, 2013; EU VRAR, 2007; ATSDR, 2004). Copper sulphate and chloride produced no mutations in Salmonella strains TA98, TA100, TA102, TA1535 and TA1537 with or without metabolic activation, even at cytotoxic concentrations or at the limit of solubility. A lack of response was also reported up to cytotoxic concentrations without metabolic activation in the SOS Chromotest (Escherichia coli PQ37), in E. coli WP2, in rec assays with Bacillus subtilis (H17 and M45), in a test for streptomycin independence in E. coli Sd4-73 and in tests for penicillin or streptomycin resistance in Micrococcus aureus FDA209 (ATSDR 2004, Greim 2004, WHO 1998).

Several genetic toxicity studies with copper sulfate showed that there were no significant increases in the occurrence of reverse mutations in tester strains of Salmonella typhimurium TA 102 (Marzin and Phi 1985), TA98 and TA100 (Moriya et al., 1983). Attention was given to one unpublished study (Ballantyne, 1994, cited in the EU VRAR, 2007), which was made available for the preparation of the Voluntary Risk Assessment Report for inorganic copper compounds. This study is the most reliable according to EU VRAR (2007). In the study, copper sulphate was tested in duplicate experiments in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in the presence and absence of metabolic activation (rat liver S-9), at doses up to 1000 µg/plate. The highest concentration caused cytotoxicity. In the second experiment, a one-hour pre-incubation step was employed. In all strains, copper sulphate failed to cause a dose-dependent, significant increase in revertant colonies over the control level, with or without metabolic activation. This study, which was conducted according to EU Annex V Test Guidelines (method B.14), clearly shows that copper sulphate is negative in the Ames test.

Similar negative findings have also been reported for copper chloride. Negative results were reported for copper chloride (CuCl2 x 2H2O) in the Ames tests, using Salmonella typhimurium TA100 and LT2 (Tso and Fung, 1981), TA98, TA102, TA1535 and TA1537 (Wong, 1988) and in TA98, TA100 and in E.Coli WP2 mutagenicity test (Codina et al., 1995). In two studies, negative results were reported for copper (I) chloride and copper (II) chloride in the Bacilis subtilis recombination assay, using strains H17 and M45 (Kanematsu et al, 1980; Nishioka, 1975). The negative result was also reported in Saccharomyces cerevisiae (Singh 1983, cited in ATSDR, 2004). In a reliable in vitro genotoxicity study copper monochloride was negative in a bacterial reverse mutation test with Salmonella typhimurium strains (TA 98, TA 100, TA 1535, and TA 1537) and with Escherichia coil (strain WP2 uvrA) with and without S9 mix at concentrations of up to 1,000 μg/plate (SIDS, 2005).

The overwhelming weight of evidence indicates that copper sulphate and copper chloride are negative in vitro in bacterial cell reverse mutation assays, and in several other bacterial cell assays up to and including cytotoxic doses (1000-~3000 μg/plate).

Justification for classification or non-classification

All available genetic toxicity studies in vitro in bacterial cells and yeasts conducted with copper gluconate, other gluconates and their derivatives as well as with inorganic copper compounds were negative. Additionally, in vivo studies conducted with glucono-delta-lactone and sodium gluconate did not induce chromosomal aberrations in mice, clear showing that no genetic toxicity can be attributed to glucoheptonate ion. Based on these data, Copper glucoheptonate can be considered as not mutagenic.

According to European Regulation (EC) No 1272/2008, copper glucoheptonate does not need to be classified and labelled as mutagenic.