Zinc EDDHA

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Chromosomal aberrations in bone-marrow cells of mice given a normal or a calcium-deficient diet supplemented with various heavy metals

Author:

Deknudt and Gerber

Year:

1979

Bibliographic source:

Mutation Research, 68 (1979) 163-168

Materials and methods

Test guideline

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:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Zinc chloride

EC Number:

231-592-0

EC Name:

Zinc chloride

Cas Number:

7646-85-7

Molecular formula:

Cl2Zn

IUPAC Name:

zinc dichloride

Test animals

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 LD50 (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.

Administration / exposure

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:

30 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm (nominal)

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: in mice with normal diet

Test results 2

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: in mice with calcium deficient diet

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 not significantly increased in mice kept on a normal diet and treated with zinc.

Any other information on results incl. tables

Treatment and diet	Body weight (g)	Serum calcium (mg/100ml)	Cells with structural aberrations	Type and Chromatid Gaps
				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

Applicant's summary and conclusion

Conclusions:

The present experiments confirm that zinc can cause severe chromosomal anomalies in animals kept on a low calcium diet. The number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.

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 number of dicentrics as well as the number of cells carrying structural aberrations was not significantly increased in mice kept on a normal diet and treated with zinc.