Zinc EDDHA

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

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

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:

in vitro mammalian chromosome aberration test

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:

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)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

L5178Y TK +/- mouse lymphoma assay: 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 varying 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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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 1242:125 was 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

Negative solvent / vehicle controls:

no

True negative controls:

no

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

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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
H2O control	61±1	146±9	0.8	-	H20 control	62±4	143±8	0.9	-
H2O 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.

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of zinc in 4 short-term mutagenicity assays

Author:

E.D. Thompson, J.A. McDermott, T.B. Zerkle, J.A. Skare, B.L.B. Evans and D.B. Cody

Year:

1989

Bibliographic source:

Mutation Research, 223 (1989) 267-272

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Zinc di(acetate)

EC Number:

209-170-2

EC Name:

Zinc di(acetate)

Cas Number:

557-34-6

Molecular formula:

C2H4O2.1/2Zn

IUPAC Name:

zinc diacetate

Specific details on test material used for the study:

Zinc acetate was purchased from Fisher Scientific, Springfield, NJ.

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

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).

Results and discussion

Test results

Test results 1

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 3

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 4

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Test results 5

Species / strain:

S. typhimurium TA 1538

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

Ames test was performed with zinc acetate. The plate-incorporation assay was performed with Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. 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.