Reaction products of sodium glucoheptonate with zinc sulfate and sodium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

To address the endpoint genetic toxicity, read-across on gluconates and derivatives and zinc compounds was performed within the frame of a weight-of-evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

Ames test

Sodium gluconate, glucono-delta-lactone and calcium gluconate

The substances were tested similar to OECD Guideline 471 on Saccharomyces cerevisiae (strain D4) and Salmonella typhimurium (3 strains) with and without metabolic activation. None of the test substances showed mutagenicity on the strains tested (SIDS, 2004).

Zinc sulphate

Zinc sulphate was negative in two tester strains Salmonella typhimurium TA97 and TA102 with and without metabolic activation (0-10 mg/plate) (Fujita et al. 1988).

Zinc salts

Zinc salts were negative in E. coli strains WP2, WP2 uvrA and WP2 exrA without metabolic activation (Venitt and Levy, 1974).

Zinc acetate

Zinc acetate was neither toxic nor mutagenic to any of the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA1538 when tested over a dose range of 50-7200 µg/plate with and without metabolic activation (Thompson et al. 1989).

Zinc 2,4-pentanedionate

When zinc was complexed with an organic ligand, 2,4-pentanedione, mutagenic results were obtained with strains TA1538 and TA98, and toxicity was obtained with both strains at 400 µg/plate (an approx. 75% reduction in bacterial lawn) (Thompson et al. 1989).

All in all, gluconates are clearly negative in Ames test (SIDS, 2004). This shows that no genetic toxicity can be attributed to gluconate and glucoheptonate ions.

Zinc was shown to be negative in Ames test when applied as zinc salts (Fujita et al. 1988, Venitt and Levy 1974, Thompson et al. 1989).

The positive response to 2,4 -pentanedione in Ames test is explained by the fact that the stable complex of zinc-pentanedionate complex prevents the binding of zinc to medium components. Zinc is transported in the cell as the organic pentanedionate complex and released after subsequent metabolism within the cell (Thompson et al. 1989). Therefore, the intracellular zinc concentration after treatment with zinc 2,4 -pentanedionate may be higher than when using other zinc compounds.

As a conclusion, no Ames mutagenicity can be expected of the glucoheptonate moiety of zinc glucoheptonate. Zinc ion is also considered to be negative in Ames test due to evidence of 3 studies covering all tester strains required according to OECD TG 471.

Mouse Lymphoma Assay

Zinc chloride

Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells without metabolic activation when applied at concentrations between 1.21 and 12.13 µg/mL (Amacher and Paillet, 1980).

Zinc acetate

Zinc acetate was genotoxic in mouse lymphoma cells with and without metabolic activation when applied at concentrations between 1.3 and 13 µg/mL (Thompson et al., 1989)

The two studies available for the mammalian gene mutation of zinc revealed conflicting results. Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells (Anmacher and Paillet, 1980), whereas Zinc acetate was shown to be genotoxic in the same cells (Thompson et al., 1989). The applied doses and the test conduct were similar in both studies.

This discrepancy is explained by the different forms of zinc tested. The ZnCl2 tested by Amacher and Paillet will dissociate and likely be bound to serum proteins, whereas a significant portion of the zinc acetate may be available for transport (Thompson et al., 1989). Therefore, zinc acetate as a test substance will possibly lead to a higher intracellular concentration of elemental zinc inducing a higher rate of gene muations. The concentrations tested by Anmacher and Paillet (1980) and Thompson et al. (1989) correspond to concentrations of 20.7 and 22.5 µg/mL when converted to the target substance zinc glucoheptonate under consideration of the purity and the molecular weight.

Chromosome aberration

Zinc acetate

Zinc acetate induced chromosome aberrations in Chinese Hamster Ovary cells in the presence and absence of metabolic activation when applied at concentrations between 25 and 80 µg/mL. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form (Thompson et al., 1989)

Zinc chloride

Zinc chloride induced severe chromosome aberrations (dicentrics) in human lymphocytes without metabolic activation at a concentration of 5x10E-5mol/L, which represents 100 times less than the toxic dose. It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the concentrations reported in the blood of people professionally contaminated by zinc

(Deknudt and Deminatti, 1978)

Zinc chloride and zinc acetate were shown to be clastogenic in two different studies on CHO cells (Thompson et al., 1989) and human lymphocytes (Deknudt and Deminatti, 1978) . Also in WHO (2001) and ATSDR (2005) stated that there are indications of some weak clastogenic effects following zinc exposure. However, genotoxicity studies conducted in a variety of test systems have failed to provide evidence that zinc is mutagenic (WHO, 2001; ATSDR, 2005).

The concentrations tested by Thompson et al. (1989) and Deknudt and Deminatti (1978) correspond to concentrations of 76.5 and 12.4 µg/mL when converted to the target substance zinc glucoheptonate under consideration of the purity and the molecular weight.

Unscheduled DNA synthesis

Zinc acetate

No UDS or toxicity was produced in rat hepatocytes by zinc acetate over a range of 10-1000 µg/mL (Thompson et al., 1989).

2,4-pentandionate

No UDS or toxicity was produced in rat hepatocytes by 2,4-pentandionate over a range of 10-1000 µg/mL (Thompson et al., 1989).

As a conclusion, zinc ion of zinc glucoheptonate is not expected to induce unscheduled DNA-synthesis.

strand damage

Zinc provides partial protection against cadmium-induced DNA damage (Coogan et al. 1992)

Conclusion

The in vitro tests had conflicting results. The reason for the conflicting results may be the form of zinc tested and the possible interactions between medium compounds and the zinc forms. For instance, when inorganic zinc (ZnCl2 or ZnSO4) is added to growth medium, the zinc is completely dissociated and may be bound by constituents in the medium (i.e. proteins). Inorganic zinc bound to medium components is unavailable for transport into the cell. On the other hand, when zinc is tested as an organic complex, there exists a competition between that complex and medium constituents for the zinc. If the organic compound binds zinc with a higher affinity than the medium components, the zinc may be transported as the organic complex. Subsequent metabolism within the cell would then release the zinc. Thus, the presence or absence of a mutagenic response in a short-term assay would depend on the type of medium, the type of cell, and the binding constant of the metal complex (Thompson et al. 1989).

Zinc is a co-factor in over 200 enzyme systems and is involved in stabilizing DNA by binding to the phosphate portions (Eichhorn and Shin, 1968). The protective effect of zinc against cadmium-induced DNA strand breaks on has also been shown by Coogan et al. (1992). Thus zinc is an essential element for cells, stabilizing the DNA. Therefore, genotoxicity of zinc does only occur at overload conditions. This may lead to a cellular stress response and to the generation of reactive oxygen species. Therefore, the generation of reactive oxygen species may the primary mechanism of action of zinc with regard to genotoxicity.

With regard to the target substance zinc glucoheptonate, it has to be considered that it is a dimeric complex with a high molecular weight of 619 g/mol and a low logP of -16.2. Therefore, in its complexed form, it is unlikely to pass the cell membrane. Assuming a dissociation of the complex, an extremely high amount of zinc glucoheptonate would be required to release a toxic concentration of elemental zinc.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

E.coli WP2 strains were grown overnight in nutrient broth at 37° C and diluted 1:50 in enriched M9 medium. Cells were collected during mid-logarithmic phase. 5 x 10E7 bacteria were spread on agar plates containing M9 medium, 0.4% (w/v) casamino-acids and 1 µg/mL L-trypophan. Test compounds were dissolved in deionised water, filter-sterilised, and 5 µL applied to the centre of each plate. Pre-existing revertants were assayed on agar plates containing M9 medium. Plates were incubated at 37° C and the maximum yield of mutants (that is, revertants to tryptophan prototrophy) was obtained 4-5 d after plating.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

not specified

Target gene:

trpE locus

Species / strain / cell type:

E. coli WP2

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

E. coli WP2

Remarks:

exrA

Metabolic activation:

without

Test concentrations with justification for top dose:

not specified

Vehicle / solvent:

deionised water, filter-sterilised

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

- Cell density at seeding (if applicable): 5x10E7

DURATION
- Preincubation period: overnight
- Exposure duration: 4-5 days

Statistics:

t-test

Species / strain:

E. coli WP2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Species / strain:

E. coli WP2

Remarks:

E. coli WP2 exrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Conclusions:

Zinc salts were negative in three different E. coli strains.

Executive summary:

The mutagenicity of zinc salts on three different E. coli strains was tested in a bacterial reverse mutation assay on agar plates. Zinc salts were negative in all three strains tested.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The Salmonella plate-incorporation assay was performed with strains TA1535, TA100, TA1537, TA1538 and TA98 as described by Maron and Ames (1983). Toxicity was estimated by measuring the extent of reduction of the bacterial lawn in the overlay agar with strain TA100.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Zinc acetate and zinc complexed with the organic ligand 2,4-pentanedionate; Zinc acetate was purchased from Fisher Scientific, Springfield, NJ. Zinc 2,4-pentanedionate was purchased from Alfa Products, Danvers, MA. 2,4-pentanedione was purchased from Aldrich, Milwaukee, WI

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 from Sprague- Dawley rats was purchased from Microbiological Associates, Bethesda, MD 20816. Aroclor 1254-induced rat liver S9 was prepared in 0.15 M KCl

Test concentrations with justification for top dose:

20 - 7200 µg/plate

Vehicle / solvent:

medium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

The plate-incorporation assay was performed with strains TA1535, TA100, TA1537, TA1538 and TA98 as described by Maron and Ames (1983).

Species / strain:

S. typhimurium TA 1535

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Remarks:

zinc complexed with 2,4-pentanedionate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Remarks:

zinc complexed with 2,4-pentanedionate

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Remarks:

zinc complexed with 2,4-pentanedionate

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

The results of the Salmonella/mammalian microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 50-7200 µg/plate.

However, when zinc was complexed with an organic ligand, 2,4-pentanedione, mutagenic results were obtained with strains TA1538 and TA98, and toxicity was obtained with both strains at 400 µg/plate (an approx. 75% reduction in bacterial lawn).

Conclusions:

The negative response with zinc acetate in the Salmonella assay suggests that binding of zinc by acetate (log of binding constant = 1.57 Martell and Smith, 1977) is either not sufficiently strong to compete with the agar medium or that zinc acetate does not cross the bacterial cell wall. The zinc 2,4-pentanedione complex (log of binding constant = 5.06) appears to be sufficiently stable to retain the zinc in the agar medium and is transported across the bacterial cell wall. The contention that the mutagenicity of zinc 2,4- pentanedione is due to zinc is supported by other studies.

Executive summary:

Ames test was performed with zinc acetate and zinc 2,4-pentanedione. While zinc acetate was neither toxic nor mutagenic in any of the tested strains, zinc 2,4 -pentanedione complex was mutagenic and cytotoxic in strains TA1538 and TA98. The negative response to zinc acetate is explained by the fact that acetate (log of binding constant = 1.57 Martell and Smith, 1977) is either not sufficiently strong to compete with the agar medium or that zinc acetate does not cross the bacterial cell wall. The zinc 2,4-pentanedione complex (log of binding constant = 5.06), on the other hand, appears to be sufficiently stable to retain the zinc in the agar medium and is transported across the bacterial cell wall. The contention that the mutagenicity of zinc 2,4- pentanedione is due to zinc is supported by other studies. As a result, zinc is mutagenic in Ames test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In each assay, cells were treated in suspension with the test material or test material plus S-9 mix for 4 hr at 37°C with constant, gentle agitation. The toxicity assay consisted of a determination of the effects of a wide range of test material on the plating efficiency of CHO cells following a 4-hr exposure with and without S-9. In the cytogenetic assay, the cells were washed following exposure to test material, seeded into T25 culture flasks, and incubated approximately 16 hr at 37°C. Cultures were then exposed to 2 µg/mL colcemid for 2 hr to arrest cells in metaphase. Mitotic cells were selectively dislodged by striking the side of the culture flask with the palm of the hand. Suspended cells were collected by centrifugation, resuspended in 0.075 M KCl and incubated 4 min at room temperature. Cells were again collected by centrifugation and fixed by slowly resuspending them in Carnoy's fixative (methanol:glacial acetic acid, 3:1). Cells were then centrifuged and resuspended in fresh fixative and stored overnight at 4°C. The following day, the fixed cells were centrifuged, resuspended in Carnoy's fixative, dropped on an inclined microscope slide, and dried. Slides were then coded and scored for chromosome number and aberrations. In each experiment the top three doses showing less than 90% relative toxicity were scored. A total of 50 metaphase spreads per dose were evaluated.

GLP compliance:

no

Type of assay:

other: Chromosome aberration in CHO cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells were obtained from the American TIype Culture Collection and were maintained in Ham's F12 medium (GIBCO) supplemented with 10% (v/v) fetal bovine serum (Reheis).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 from Sprague- Dawley rats was purchased from Microbiological Associates, Bethesda, MD 20816. Aroclor 1254-induced rat liver S9 was prepared in 0.15 M KCl

Test concentrations with justification for top dose:

without metabolic activation: [µg/mL] 25, 34, 45
with metabolic activation: [µg/mL] 45, 60, 80

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 16 h
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 40 h

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):
2 µg/mL colcemid

STAIN (for cytogenetic assays):
Carnoy's fixative (methanol:glacial acetic acid, 3:1)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The following day, the fixed cells were centrifuged, resuspended in Carnoy's fixative, dropped on an inclined microscope slide, and dried.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
A total of 50 metaphase spreads per dose were evaluated

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

zinc acetate

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-dependent positive responses were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity.

			Aberration types
Dose(µg/ml)	Metabolic activation	Relative cloning efficiency	Chromatid^bgaps	Chro­matid break	Frag­ments	Exchanges	Rings	>10 Aber­rations	%Poly­ploidy	Aberrations^cper cell
Neg. Control (water)	—	100 (58)^a	2	0	0	0	0	0	2	0
25	—	100	2	0	0	0	0	0	4	0
34	—	36	1	5	1	15	0	0	4	0.42
45	—	53	3	10	5	22	0	5	4	1.74
Pos. control(TEM-0.5 Mg/ml)	—	2	4	23	26	42	1	8	4	3.44
Neg. control (water)	+	100(33)^a	0	0	0	0	0	0	0	0
45	+	73	3	7	0	10	0	1	4	0.54
60	+	76	2	17	2	21	0	3	6	1.40
80	+	58	1	7	0	5	0	5	0	1.24
Pos. control (cyclophosphamide 35 fig/ml)	+	9	9	28	17	45	0	4	4	2.60
a The cloning efficiency for water is arbitrarily set at 100. The number in parentheses is the number of clones formed when 200 cells (was determined by hemacytomer counts) were plated. The relative cloning efficiency of each dose equals the number of viable clones for that dose divided by the viable clones in the water control, times 100. b Counted, but not used in the aberrations per cell calculation. c Aberrations per cell is the total number of aberrations (excluding gaps) divided by the number of cells scored (50 for all doses). Cells with > 10 aberrations are counted as 10 aberrations.

Conclusions:

Dose-dependent positive responses were obtained in the presence and absence of the S9 activation system, although the S9 reduced both the clastogenic response and the toxicity. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form. However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Executive summary:

A chromosome aberration assay with zinc acetate was conducted in Chinese Hamster Ovary cells. Zinc acetate induced chromosome aberraions in the presence and absence of metabolic activation. These results indicate that zinc is an effective clastogen when presented to a susceptible cell population in an appropriate form. However, in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Human lymphocytes from the same healthy donor were cultured for 48 (first cellular division) or 72 h (second cellular division) in Ham’s F 10 medium to which different concentrations of zinc chloride had been added. 3 subtoxic doses of each salt (2, 10 and 100 times less than the toxic dose), were added to 48 and 72-h cultures at 0 h and 24 h after initiation. Chromosome preparations were made and 100 well spread metaphases from each culture were analysed for the presence of numerical and structural aberrations.

GLP compliance:

no

Type of assay:

other: in vitro mammalian chromosome aberration assay

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

human lymphocytes from healthy donor

Metabolic activation:

without

Test concentrations with justification for top dose:

2, 10 and 100 times less than the toxic dose: 0 M; 3x10E-4 M; 3x10E-5 M

Vehicle / solvent:

Ham’s F 10 medium

Untreated negative controls:

yes

Remarks:

no treatment

Negative solvent / vehicle controls:

yes

Remarks:

no treatment

True negative controls:

not specified

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 0 and 24 h
- Exposure duration: 24, 48, and 72 h

NUMBER OF CELLS EVALUATED:
100 cells per treated culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
100 well spread metaphases from each culture were analysed for the presence of numerical and structural aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Statistics:

A chi-square analysis was performed.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Severe aberrations such as dicentric chromosomes were recorded only in lymphocyte cultures treated with the lowest concentration of zinc chloride (3 x 10E-5) added at time 0, regardless whether the cultures were fixed after 48 or 72 h.

The most common aberration found for all tested metal salts was the occurrence of chromosome fragments. Dicentric chromosomes were only recorded in lymphocyte cultures treated with the lowest concentration of zinc chloride (3x10E-5 M) added at time 0, regardless whether the cultures were fixed after 48 or 72 h.

The chi-square analysis did not show that the incidence of dicentrics in the cells treated with zinc were significant when tested against the controls only. If, however, controls combined with the cadmium and lead exposed were tested against all zinc treated lymphocytes the difference was highly significant (P = 0.004).

Conclusions:

The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5 M) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.

Executive summary:

Chromosome aberrations in human lymphocytes induced by zinc chloride were analysed. The results suggest that high levels of zinc (3 x 10E-3 M) are cytotoxic and that lower concentration (3 x 10E-5) can cause severe chromosome aberrations (dicentrics). It must be pointed out, however, that the concentration of zinc used in the present experiments are extremely high representing up to 1000 times the respective concentrations reported in the blood of people professionally contaminated by heavy metals and which have been shown to be 22.4 µg% for zinc.

Reference 5

Endpoint:

genetic toxicity in vitro, other

Remarks:

DNA single strand damage

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The alkaline elution technique was used to assess DNA single strand damage (SSD) in cultured cells derived from rat hepatocytes (TRL-1215), a cell line previously shown to have an active metallothionein (MT) gene. The ability to detect SSD in TRL-12 15 was established following exposure to gamma-irradiation.

Briefly, the DNA of TRL- 1215 cells was labeled with [14C] thymidine by growing cells for two doublings (ca. 48 hr) in the presence of 0.02 µCi/mL [14C]thymidine. Following labeling and prior to treatment, cells were pulsed for 1 hr with 2 mM unlabeled thymidine. After treatment, 5 x 10E5 cells were loaded per filter with duplicate filters run for each group. Cells were lysed at room temperature by the addition of 2.5 ml of lysis solution containing 2% SDS and 25 mM disodium EDTA (pH 9.6). After 30 min, the solutions were filtered by gravity and each filter was washed with 1.0 ml 25 mM EDTA (pH 9.6). DNA was eluted in the dark (35 µL/min) using a solution containing 25 mM EDTA adjusted to pH 12.1 with tetraethyl ammonium hydroxide. Fractions were collected at 90-min intervals for 12 hr. Following elution, each pump line was washed with 5 ml 0.4 N NaOH. The DNA content of filters, fractions, and washes was determined isotopically. Strand scission factor (SSF) was calculated as described by Sugiyama (1986) using the equation SSF = log(A/B), where A equals the 6-hr fraction retained for the control, nonexposed cells and B equals the 6-hr fraction retained for the treated cells. Rad equivalents were calculated using the linear relationship established between SSF and dose of gamma-irradiation.

GLP compliance:

no

Type of assay:

other: DNA strand damage-alkaline elution technique

Specific details on test material used for the study:

Zn(C2H3O2)2.2H2O

Species / strain / cell type:

mammalian cell line, other: TRL-1215

Remarks:

The TRL-1215 cell line is an epithelial-like cell line derived from the livers of 10-day-old Fischer 344 rats (Idoine et al.. 1976). These cells are diploid and nontumorigenic at the passage levels used in these studies.

Details on mammalian cell type (if applicable):

Cells were grown using William’s D medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/mL streptomycin and maintained in a humidified atmosphere of 5% C02/95% air at 37°C.

Metabolic activation:

without

Test concentrations with justification for top dose:

80 µM

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

TRL-1215 cells were pretreated with zinc by exposing monolayer cultures in medium to 80 µM zinc acetate 18 hr prior to isolation.

Species / strain:

mammalian cell line, other: TRL-1215

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

zinc acetate decreased the cytotoxicity of cadmium

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Cells treated with 80 µM zinc acetate do not induce increased strand damage when compared to the solevent control. The amount of DNA damage is even reduced in cells treated with zinc acetate in comparison to untreated cells. Furthermore, pre-treatment with zinc acetate decreases the amount of DNA strand breaks induced by cadmium (figure 4). In addition, pretreatment of cells with 80 µM zinc acetate decreases cytotoxicity of cadmium, significantly (p < 0.05). Besides, an 18-h exposure to 80 µM zinc acetate resulted in an about 11-fold increase in metallothionein (MT) protein levels as compared to nontreated cells (figure 5).

Conclusions:

Zinc provides partial protection against cadmium-induced DNA damage, although the exact mechanism is unknown. It is reasonable to assume that metallothionein (MT) plays a role in this protection. It has been shown in this study that an 18-h exposure to 80 µM zinc acetate resulted in an about 11-fold increase in MT protein levels as compared to nontreated cells. MT has been reported to serve as a radical scavenger (Thornalley and Vasak, 1985; Abel and Ruiter, 1989). Thus, excess MT could possibly protect DNA from cadmium generated radicals.

Executive summary:

The effect of zinc pretreatment on cadmium-induced DNA strand damage was determined. The alkaline elution technique was used to assess DNA single strand damage (SSD) in cultured cells derived from rat hepatocytes (TRL-1215). Zinc provides partial protection against cadmium-induced DNA damage, although the exact mechanism is unknown. It is reasonable to assume that metallothionein (MT) plays a role in this protection. It has been shown in this study that an 18-h exposure to 80 µM zinc acetate resulted in an about 11-fold increase in MT protein levels as compared to nontreated cells. MT has been reported to serve as a radical scavenger (Thornalley and Vasak, 1985; Abel and Ruiter, 1989). Thus, excess MT could possibly protect DNA from cadmium generated radicals.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Zinc chloride was tested for the potential to induce trifluorothymidine-resistant (TFT Res) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses for 3 h. 48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media and plated. Trifluorothymidine resistance (TFT Res) was determined by adding 4 µg/ml TFT to one set of plates. All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 after incubation at 37°C. Those TFT Res colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants.

GLP compliance:

no

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Specific details on test material used for the study:

ZnCl2.6H2O

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK +/- 3.7.2C (Clive, 1973) cells originally obtained from D. Clive had been thawed from a frozen ampule within 1 month of the completion of these experiments. These cells were determined free of mycoplasma via 3 methods including fluorescent antibody to M. hyorhinus by an independent laboratory and were routinely suspended in R5 so as to remain in exponential growth.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

1:1.334 serial dilutions; 1.21 - 12.13 µg/mL

Vehicle / solvent:

filter-sterilized saline

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 6 x 10E6

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 9 d

SELECTION AGENT (mutation assays):
Trifluorothymidine

NUMBER OF REPLICATIONS:
3 replicate treatments for 2 cell cultures, respectively (=6 plates)

NUMBER OF CELLS EVALUATED:
All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 with a Artek model 870 bacterial Colony Counter.

DETERMINATION OF CYTOTOXICITY:
Cell survival for each culture was the product of growth in suspension culture and cloning efficiency in soft-agar medium, each relative to solvent controls.

Evaluation criteria:

Representative TFT Res colonies derived from cultures originally treated for 3 h with either cadmium chloride, nickel chloride or trans-Pt(II)(NH3)2Cl2 were removed from soft-agar plates which had been dosed with 4 pg/ml TFT 7 days prior, broken-up by vortexing, transferred to antibiotic-free Rs, then subcultured another 7 days. These presumed TK -/- cell lines were then recloned in soft-agar medium containing either 100 pg/ml bromodeoxyuridine (BUdR) or 9 pg/ml thymidine, 15 pg/ml hypoxanthine, 0.3 pg/ml methotrexate, and 22.5 µg/ml glycine (THMG) to determine BUdR resistance and THMG resistance, resp. Absolute cloning efficiencies were determined simultaneously by plating some of these same cells in corresponding soft-agar medium alone. Confirmation of the TK -/- phenotype (BUdR-resistant and THMG-sensitive) after 7 days subculture in TFT-free medium constitutes a minimum criterion of TFT Res variant stability.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Zinc chloride was not genotoxic in L5178Y mouse lymphoma cells.

Executive summary:

Zinc chloride was tested for the potential to induce trifluorothymidine-resistant (TFT Res) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses for 3 h. 48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media and plated. Trifluorothymidine resistance (TFT Res) was determined by adding 4 µg/ml TFT to one set of plates. All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 after incubation at 37°C. Those TFT Res colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. The result was negative for ZnCl2.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

L5178Y TK +/- mouse lymphoma assay: Zinc acetate was tested according to the procedures of Clive and Spector (1975) and Clive et al., 1979. Zinc acetate was tested for the potential to induce trifluorothymidine-resistant (TFT Res) mutants in L5178Y/TK+/- mouse lymphoma cell by directly exposing cells to varied doses for 3 h. 48 h after treatment, metal-treated cells and solvent controls were cloned in soft-agar media and plated. Trifluorothymidine resistance (TFT Res) was determined by adding 4 µg/ml TFT to one set of plates. All colonies growing either in the presence of TFT (TFT Res) or its absence (viable count colonies) were counted on day 7 after incubation at 37°C. Those TFT Res colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants.

GLP compliance:

no

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells were received from D. Clive, Burroughs, Welcome Co., Research Triangle, NC

Metabolic activation system:

Aroclor-induced rat liver S9 from Sprague- Dawley rats was purchased from Microbiological Associates, Bethesda, MD 20816. A 2:1 mixture of Aroclor 1242:125was used to induce rat livers for the mouse lymphoma assay. This S9 was prepared in 0.25 M sucrose.

Test concentrations with justification for top dose:

[µg/mL] 1.3, 1.8, 2.4, 3.2, 4.2, 5.6, 7.5, 10, 13

Vehicle / solvent:

medium

Untreated negative controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

according to the procedures of Clive and Spector (1975) and Clive et al. (1979).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the TK +/- mouse lymphoma assay with zinc acetate are shown in Table 1. Dose-dependent positive responses were obtained in the presence and absence of the S9 metabolic activation system with a doubling of the mutation frequency occurring at 10 µg/ml for both portions of the assay (Table 1).

Table 1: Results of the L5178Y TK+/- Mouse Lymphoma Assay on Zinc acetate

Without S9 activation					With S9activation
Concentration (µg/ml)	Colonies per TFT plate	Colonies per VC plate	Mutation frequency per 10^4surviving cells	% total growth	Concentration (µg/Plate)	Colonies per TFT plate	Colonies per VC plate	Mutation frequency per 10^4surviving cells	total growth
H20 control	61±l	146±9	0.8	-	H20 control	62±4	143±8	0.9	-
H20 control	68±7	127±15	1.1	-	H20 control	38±1	145±11	0.5	-
1.3	59±1	138±5	0.9	79	4.2	63±4	162±5	0.8	Ill
1.8	61±6	113±3	1.1	61	5.6	92±7	169±6	1.1	112
2.4	62±6	152±5	0.8	113	7.5	87±7	151±6	1.2	79
3.2	59±1	151±11	0.8	119	10	78±3	132±21	1.2	74
4.2	59±1	133±5	0.9	82	13	94±4	159±10	1.2	79
5.6	60±3	152±5	0.8	130	18	108±8	130±14	1.7	49
7.5	54±14	144±14	0.8	82	24	194±7	138±10	2.8	33
10	117±14	127±5	1.8	60	32	275±12	135±8	4.1	19
13	194±12	92±2	4.2	31	42	230±2	62±5	7.4	8
Pos. control EMS					Pos.control DMBA
(0.5µg/ml)	251±19	80±3	6.3	35	(5.0µg/ml)	145±1	132±4	2.2	64
(1.0µg/ml)	142±7	17±2	16.7	4	(7.5µg/ml)	194±6	125±13	3.1	57

Conclusions:

The results presented here indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Executive summary:

A mouse lymphoma TK+/- assay was perormed with zinc acetate. The results indicate zinc is an effective mutagen when presented to a mouse lymphoma cells in an appropriate form. However, controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Unscheduled DNA synthesis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Unscheduled DNA synthesis (UDS) in rat liver primary cell cultures: After exposed to the test chemical UDS was demonstrated autoradiographically by grains over the nuclei following exposure to [3H]TdR.

GLP compliance:

no

Type of assay:

other: Unscheduled DNA synthesis in rat hepatocytes

Specific details on test material used for the study:

zinc acetate and 2,4-pentanedione;
Zinc acetate was purchased from Fisher Scientific, Springfield, NJ. Zinc 2,4-pentanedionate was purchased from Alfa Products, Danvers, MA. 2,4-pentanedione was purchased from Aldrich, Milwaukee, WI

Species / strain / cell type:

primary culture, other: rat hepatocytes

Metabolic activation:

without

Test concentrations with justification for top dose:

10 - 1000 µg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 18 to 24 h

-exposure to test chemical
-exposure to [3H]TdR (tritiated thymidine)

Species / strain:

primary culture, other: rat hepatocytes

Remarks:

zinc acetate

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

primary culture, other: rat hepatocytes

Remarks:

zinc 2,4-pentanedionate

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Neither form of zinc induced UDS or produced toxicity in these cells over a range of 10-1000 µg/mL.

Conclusions:

Neither form of zinc induced unscheduled DNA synthesis or produced toxicity in these cells over a range of 10-1000 µg/ml

Executive summary:

An unscheduled DNA synthesis assay was performed with zinc acetate and zinc 2,4 -pentanedionate in a primary cell culture of rat hepatocytes. Neither form of zinc induced unscheduled DNA synthesis or produced toxicity in these cells over a range of 10-1000 µg/ml. This indicates that no DNA damage was induced by zinc acetate or zinc 2,4 -pentanedionate.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate in organic fluids releasing their metals that can further interact with a variety of biomolecules, while no genetic toxicity can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, producing adverse genotoxic effects. Therefore, data on genetic toxicity potential of several organic and inorganic zinc compounds have been taken into account to address genetic toxicity potential of zinc cation which could be released from the zinc glucoheptonate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The zinc glucoheptonate content in the registered product is 75 %. Another component is Na2SO4. Sodium is a macroelement occurring in surface waters and in living organisms in considerable amounts. Sulfur species are also found in living organisms. Thus, these cations and anions are considered not to impact the mutagenicity of zinc to mammals.

The purity of zinc sulphate used in this study is not specified.

3. ANALOGUE APPROACH JUSTIFICATION
Since glucoheptonate anion is considered not mutagenic to bacterial strains, certain mutagenicity potential, if any, could be attributed to zinc. Therefore, data on mutagenicity of inorganic zinc compounds are used to address mutagenicity of zinc.

4. DATA MATRIX
please refer to the detailed data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Results of Mutation Test with magnesium dietary supplements

Dose (mg/plate)	No of Revertants /plate
Magnesium Oxide, Heavy
	TA97		TA102
	-S9	+S9	-S9	+S9
10	111	148	216	469
5	108	185	193	461
1	103	187	183	460
0.5	101	182	179	425
0.1	119	178	192	445
0	118	179	205	461
Positive control	178	3,987	3,576	1,820
Magnesium Carbonate, Basic, Heavy
	-S9	+S9	-S9	+S9
1	123	160	227	419
0.5	114	167	219	454
0.1	103	166	210	391
0.05	98	182	192	440
0.01	100	169	234	403
0	115	175	245	416
Positive control	175	1,997	3,187	2,112
Solvent: DW (not specified)

Conclusions:

zinc sulfate was negative in two tester strains Salmonella typhimurium TA97 and TA102. This is also expected for zinc glucoheptonate.

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. Zinc sulfate was negative in two tester strains.

Based on the read-across hypothesis, no genetic toxicity is expected for zinc glucoheptonate.

Reference 10

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: summary of mutagenicity data

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented peer-reviewed report.

Qualifier:

no guideline available

Principles of method if other than guideline:

Summary of genetic toxicity study results conducted with monosaccharides, disaccharides, and related ingredients as used in cosmetics.

GLP compliance:

not specified

Type of assay:

other: summary

Species / strain:

other: The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are overwhelmingly negative.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

other: not applicable (summary result)

Untreated negative controls validity:

other: not applicable (summary result)

Positive controls validity:

other: not applicable (summary result)

Conclusions:

The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are negative

Executive summary:

Summary of genetic toxicity study results conducted with monosaccharides, disaccharides, and related ingredients as used in cosmetics.

The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are negative.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: summry of available in vitro and in vivo genetic toxicity data

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented peer-reviewed report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study was made on one yeast strain : Saccharomyces cerevisiae, strain D4 and 3 bacteria strains: S. typhimuriumTA1535, TA1537 and TA 1538. Positive controls are different from those in the OECD 471; only 3 concentration tested.

Principles of method if other than guideline:

Summary of genetic toxicity data on glucono-delta-lactone, sodium or calcium gluconate

GLP compliance:

not specified

Type of assay:

other: summary of a variety of data

Target gene:

his-

Species / strain / cell type:

other: S.typhimurium TA 1535, TA 1537, TA 1538

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

strain D4

Metabolic activation:

with and without

Metabolic activation system:

The tissue homogenates and supernatants (9000 g) were prepared from tissues of mouse (ICR random bred adult males); rat (Sprague-Dawnley adult males) and monkey (Macaca mulatta adult males).

Test concentrations with justification for top dose:

Sodium gluconate: 0.06, 0.012, 0.024 µg/mL (Salmonella typhimurium); 12.5, 25 and 50 µg/mL (yeast);
Glucono-delta-lactone: 2.5, 5 (5 µg/mL plate test; Salmonella typhimurium); 12.5 and 25 µg/mL (yeast);
Calcium gluconate: 12.5, 25 and 50 µg/mL (Salmonella typhimurium); 7.5, 15 and 30 µg/mL (yeast).

Vehicle / solvent:

- Solvent used: 0.067 M phosphate buffer, pH 7.4

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Remarks:

without S9

Positive control substance:

2-nitrofluorene

ethylmethanesulphonate

other: Quinacrine or quinacrinemustard (QM)

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Remarks:

with S9

Positive control substance:

2-acetylaminofluorene

N-dimethylnitrosamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and in suspension

DURATION
- Exposure duration:
- Glucono-delta-lactone: 4 days: bacteria and yeasts (plate test); 4 hours (yeasts) and 1 hour (bacteria) in suspension test.
- Sodium gluconate: 48 to 72 hours bacteria and yeasts (plate test); 4 hours (yeasts) and 1 hour (bacteria) in suspension test.
- Calcium gluconate: 4 days: bacteria and yeasts (plate test); 4 hours (yeasts) and 1 hour (bacteria) in suspension test.

DETERMINATION OF CYTOTOXICITY
- Glucono-delta-lactone: 50% survival in bacteria calculated was at 1% (10 μg/mL) test substance and 5% (50 μg/mL) for yeast;
- Sodium gluconate: 50% survival in bacteria calculated was at 0.0024 % test substance and 5% for yeast;
- Calcium gluconate: 50% survival in bacteria calculated was at 5.00 % test substance and 3.00% for yeast.

Tests in suspension without S9 mix: Bacterial plates were scored after incubation for 48 hours at 37°C. The yeast plates were incubated at 30°C for 3-5days before scoring.

Species / strain:

S. typhimurium, other: TA 1535; TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Cytotoxic concentration (50% survival) (μg/ml):

Sodium gluconate: 0.024 (bacteria), 50 (yeast);

Glucono-delta-lactone: 10 (bacteria), 50 (yeast);

Caclium gluconate: 50 (bacteria), 30 (yeast).

Conclusions:

The available in vitro mutagenicity data with glucono-delta-lactone, sodium or calcium gluconate were negative.

Executive summary:

Sodium gluconate, glucono-delta-lactone and calcium gluconate were tested on Saccharomyces cerevisiae and Salmonella typhimurium with and without metabolic activation. 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) with and without metabolic activation. Only 3 concentrations were tested where OECD guideline recommends at least 5 concentrations. None of the test substances showed mutagenicity on the strains tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

To address the endpoint genetic toxicity read-across on gluconates and derivatives and zinc compounds was performed within the frame of a weight of evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

Chromosome aberration

Data on Gluconates

Glucono-delta-lactone and sodium gluconate did not induce chromosomal aberrations in mice (CIR, 2014). 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 (CIR, 2014). This shows that no genetic toxicity can be attributed to gluconate and glucoheptonate ions.

Data on zinc chloride (Deknudt and Gerber, 1979)

Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50/30 days. After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc.

The present experiments confirm that zinc can cause severe chromosomal anomalies in animals kept on a low calcium diet. No significant increase in the number of chromosome aberrations was induced by zinc in animals receiving non calcium-deficient diet. This is explained by the fact that calcium fulfils an essential role in maintaining the structure of chromosomes and, in its absence, other similar ions may take its place leading to a perturbation in the DNA--histone bonds and possibly causing chromosome breaks and/or interfering with repair processes (Deknudt and Gerber, 1979).

Zinc homeostasis in mammals

As described in EU RARs (2004) within certain limits, mammals can maintain the total body zinc and the physiologically required levels of zinc in the various tissues, constant, both at low and high dietary zinc intakes. The sites of regulation of zinc metabolism are: absorption of zinc from the gastrointestinal tract, excretion of zinc in urine, exchange of zinc with erythrocytes, release of zinc from tissue, and secretion of zinc into the gastrointestinal tract. Regulation of gastrointestinal absorption and gastrointestinal secretion most likely contributes the most to zinc homeostasis. Thus, the likelihood of zinc being genotoxic is precluded in vivo under standard feeding conditions (Thompson et al., 1989).

Conclusion

In vivo no genetic toxicity can be expected for zinc glucoheptonate. As was shown, neither glucoheptonate ions, nor elemental zinc induce chromosome aberration in mice under standard feeding conditions. In addition, mammals have effective mechanisms to regulate zinc homeostasis, and thus prevent toxic overload conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50/30 days. After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined.

GLP compliance:

no

Type of assay:

other: mammalian bone marrow chromosome aberration assay

Species:

mouse

Strain:

C57BL

Details on species / strain selection:

8-week-old male mice of the C57B1 strain, weighing about 25 g

Sex:

male

Details on test animals or test system and environmental conditions:

25 mice (8-week-old male mice of the C57B1 strain, weighing about 25 g) were maintained for one month on a standard diet (1.1 % calcium) or an a low-calcium diet (0.03 % calcium), to which zinc chloride (0.5 % zinc) has been added. As determined in a pilot experiment, these concentrations, added to the low-calcium diet, represent the LD 50/30 days, whereas only a few animals died in the groups receiving heavy metals added to a normal-calcium diet. Two additional groups receiving a normal or low-calcium diet served as controls. Each of the groups studied consisted of 25 mice; 10 survivors were killed for assay after one month.

Route of administration:

oral: feed

Vehicle:

no vehicle used

Details on exposure:

25 mice (8-week-old male mice of the C57B1 strain, weighing about 25 g) were maintained for one month on a standard diet (1.1 % calcium) or an a low-calcium diet (0.03 % calcium), to which zinc chloride (0.5 % zinc) has been added. As determined in a pilot experiment, these concentrations, added to the low-calcium diet, represent the LD 50/30 days

Duration of treatment / exposure:

one month

Frequency of treatment:

daily

Dose / conc.:

5 ppm (nominal)

Dose / conc.:

0.75 mg/kg bw/day

Remarks:

calculated from the test concentration in ppm using the default conversion factor for mice of 0.15 (Guidelines for the preparation of toxicological working papers for the Joint FAO/WHO Expert Committee on Food Additives, Geneva, December 2000)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Positive control(s):

no positive control available

Tissues and cell types examined:

bone-marrow cells from both femurs

Details of tissue and slide preparation:

1 h before being killed by cervical dislocation, each mouse received an i.p. injection of 0.4 ml/30 g body weight of a 0.025% colchicine solution (p.a. from Merck). After the killing, blood was taken for calcium determination, and bone-marrow cells from both femurs were obtained by washing the shafts with a 2.2% sodium citrate solution. The cells were centrifuged, kept in hypotonic (1%) sodium citrate solution for 12 min, centrifuged again and fixed in ethanol : acetic acid (3 : 1). A few drops of the final cell suspension were spread on a clean glass slide and stained with lacto-orcein. 50 well-spread metaphases from each animal (a total of 500 from each group) were analysed for the presence of structural chromosomal aberrations.

Evaluation criteria:

not specified

Statistics:

The chromosomal data were statistically evaluated by chi-square analysis, and the weight and calcium data were tested by an analysis of variance.

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Treatment with heavy metals, as well as the low-calcium diet significantly reduced the body weight of the mice, and the effect was more pronounced when treatments with heavy metals and low calcium diet were combined. In general, the intoxicated animals on a low calcium diet had lost weight, were weak, seemed anaemic and had brittle femurs. Serum calcium was reduced in animals on a low-calcium diet, and this effect was accentuated by intoxication with heavy metals whereas this intoxication as such did not significantly influence calcium levels in animals on a normal diet. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc.
The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc.

Treatment and diet	Body weight (g)	Serum calcium (mg/100ml)	Cells with structural aberrations	Type andChromatidGaps
				Chromatid aberrations		Chromatid aberrations
				gaps	Breaks	Gaps	Fragments	Dicentrics
Control+ Ca	29.90±0.12	10.24±0.06	1.80±0.60	1.20±0.49			0.6±0.35
Control - Ca	21.80±0.27 !!	9.45±0.15 !!	2.00±0.63	1.80±0.60			0.4±0.28
Zinc+ Ca	17.90±0.23 **	9.76±0.29 *!	2.80±0.75	1.80±0.60	0.2±0.2		0.4±0.28	0.4±0.28
Zinc - Ca	12.05±0.25 **!!	8.76±0.24	5.00±1.00 **	3.20±0.80		0.4±0.28	0.6±0.35	1.2±0.49
^a All values represent means -+ standard errors (Poisson errors for counting data). ^Statistically significant differences from the respective controls without heavy metals are indicated as * and ** for the p <= 0.05 and p<= 0.01 levels. ^Differences from the respective treated group with calcium in the diet are shown as ! and !! for the p <= ^0.05 and p <= 0.01 levels ^b Exact probability 0.06

Conclusions:

The present experiments confirm that zinc can cause severe chromosomal anomalies, particularly in animals kept on a low calcium diet.

Executive summary:

Mice kept on a normal (1.1% calcium) or low-calcium (0.03%) diet were exposed for one month to zinc chloride (0.5% Zn). The concentrations, given in a poor calcium diet, represent a LD 50/30 days. After the mice were killed bone-marrow cells were assayed for chromosomal aberrations, and serum calcium was determined. The number of dicentrics as well as the number of cells carrying structural aberrations was significantly increased in mice kept on a calcium-deficient diet and treated with zinc.

The present experiments confirm that zinc can cause severe chromosomal anomalies, particularly in animals kept on a low calcium diet.

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:

weight of evidence

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;

- Concentration of test material in vehicle:
- 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 (nominal)

Remarks:

single dose administration

Dose / conc.:

4 000 mg/kg bw/day (nominal)

Remarks:

single dose administration

Dose / conc.:

8 000 mg/kg bw/day (nominal)

Remarks:

single dose administration

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

4 day repeated dose

Dose / conc.:

4 000 mg/kg bw/day (nominal)

Remarks:

4-day repeated dose

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 (= 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 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.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

- 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 (nominal)

Remarks:

single dose administration

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

single dose administration

Dose / conc.:

10 000 mg/kg bw/day (nominal)

Remarks:

single dose administration

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

4 day repeated dose

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

4-day repeated dose

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro genetic toxicity studies with Calcium gluconate, Sodium gluconate and glucono-delta-lactone

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 where 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 the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are 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 (CIR, 2014). 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 (CIR, 2014).

Based on these data, no genetic toxicity can be attributed to gluconate ions, its derivatives and other sugar-like compounds including glucoheptonate ion.

Justification for classification or non-classification

The registered substance does not have to be labelled as mutagenic substance according to European Regulation (EC) No 1272/2008, because all available genetic toxicity studies in vitro in bacterial cells and yeasts conducted with sodium gluconate or glucono-delta-lactone were negative. Also, several zinc compounds were negative in Ames test in a variety of different tester strains. The positive outcomes of the in vitro studies with zinc compounds on mammalian cells are indicative of potentiated formation of active oxygen species under overload conditions.

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 moiety. An in vivo study with zinc chloride (chromosome aberration, mice bone marrow) had positive results, but only in mice kept under calcium-deficient feeding conditions.

Taking into account these study results and considering the facts that in the target substance the zinc ion is complexed by a dimeric structure with a high molecular weight and that the zinc homeostasis in mammals is highly regulated, zinc glucoheptonate is considered to be not mutagenic.