Dizinc pyrophosphate

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

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:

study report

Title:

Unnamed

Year:

1974

Report date:

1974

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A chromosomal aberration assay was conducted in rats to evaluate the genotoxic potential of zinc sulphate. The study utilizes examination of bone marrow cells arrested in C-metaphase from rats exposed to the test compound as compared to positve and negative control animals.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Zinc sulphate

EC Number:

231-793-3

EC Name:

Zinc sulphate

Cas Number:

7733-02-0

Molecular formula:

H2O4S.Zn

IUPAC Name:

zinc sulfate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Flow Laboratories
- Age at study initiation: 12 weeks old
- Weight at study initiation: 280 to 350 g
- Assigned to test groups randomly: not specified
- Fasting period before study:
- Housing: one to five per cage
- Diet: not specified
- Water: not specified
- Acclimation period: 4 - 11 days

ENVIRONMENTAL CONDITIONS
not specified

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.85% saline

Duration of treatment / exposure:

acute study: once
subacute study: five days

Frequency of treatment:

subacute: daily

Post exposure period:

acute study: 6, 24 and 48 hours
subacute study: 6 hours after last dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.75 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

27.5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

275 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per treatment group
3 animals per control group

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: one treatment intraperitoneally, after 48 hours cells were prepared for analysis
- Doses / concentrations: 0.3 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: preliminary toxicity test: LD5 was used as highest dose, 1/10 of LD5 was used as intermediate dose and 1/100 of LD5 was used as low dose

DETAILS OF SLIDE PREPARATION: Four hours after the last compound administration, and two hours prior to killing, each animal was given 4 mg/kg of colcemid intraperitoneally in order to arrest the bone marrow cells in C-mitosis. Animals were killed by using CO2, and the adhering muscle and epiphysis of one femur were removed. The marrow "plug" was removed with a tuberculin syringe and an 18 gauge needle, aspirated into 5 mL of Hanks' balanced salt solution (BSS) in a test tube and capped. The specimens were centrifuged at 1500 rpm in a table-top centrifuge for 5 minutes, decanted, and 2 mL of hypotonic 0.5% KCl solution was added with gentle agitation to resuspend the cells. The specimens were then placed in a 37°C water bath for 20 minutes in order to swell the cells. Following centrifugation for 5 minutes at 1500 rpm, the supernatant was decanted and 2 mL of fixative (3:1 absolute methanol:glacial acetic acid) was added. The cells were resuspended in the fixative with gentle agitation, capped, and placed at 4°C for 30 minutes. The specimens were again centrifuged, decanted, 2 mL of prepared fixative was added, and the cells were resuspended and placed at 4°C overnight. The following day the specimens were again centrifuged, decanted and 0.3 - 0.6 mL of freshly prepared fixative was added to obtain a suitable density. The cells were resuspended and 2 - 3 drops of the suspension were allowed to drop onto a clean, dry slide held at 15°C from the horizontal. As the suspension flowed to the edge of the slide, it was ignited by an alcohol burner and allowed to flame. Following ignition, the slides were allowed to dry at room temperature overnigt. Duplicate slides were prepared. The slides were stained using a 5% Giemsa solution for 20 minutes, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 minutes. The slides were then mounted using Permount and 24 x 50 mm coverglasses.

METHOD OF ANALYSIS: Suitable metaphase spreads that were countable were examined. The chromosomes of each cell were counted and only diploid cells were analysed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverisation, and any other chromosomal aberrations which were observed. 50 metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Evaluation criteria:

not reported

Statistics:

not reported

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: acute study: metaphase summary sheet
Compound	Dose (mg/kg bw)	Time*	# of animals	# of cells	Mitotoic index %***	% cells with breaks	% cells with reunion	% cells other aberr.**	% cells with aberr.
Negative control	saline	6	3	150	4	0	0	0	0
		24	3	150	5	0	0	0	0
		48	3	150	7	0	0	0	0
Low level	2.75	6	5	250	5	0	0	0	0
		24	5	200	4	0	0	0	0
		48	5	250	3	0	0	0	0
Intermediate level	27.5	6	5	250	9	0	0	0	0
		24	5	250	4	0	0	0	0
		48	5	250	7	0	0	0	0
LD5	275	6	5	250	4	0	0	0	0
		24	5	250	4	0	0	0	0
		48	5	250	6	0	0	0	0
Positive control TEM	0.3	48	5	250	4	0.4	27	14(a)	41
* time of kill after injection (hours) ** cells that have polyploidy (P), pulverisation (pp), fragments (f) or greater than 10 aberrations (a). *** % of cells in mitosis: 500 cells observed/animal.

Table 2: subacute study: metaphase summary sheet
Compound	Dose* (mg/kg bw)	# of animals	# of cells	Mitotoic index %***	% cells with breaks	% cells with reunion	% cells other aberr.**	% cells with aberr.
Negative control	saline	3	150	8	0	0	0	0
Low level	2.75	5	250	5	0	0	0	0
Intermediate level	27.5	5	250	8	0	0	0	0
LD5	275	5	250	6	0	0	0	0
* Dosage 1x/day x 5 days ** cells that have polyploidy (P), pulverisation (pp), fragments (f) or greater than 10 aberrations (a). *** % of cells in mitosis: 500 cells observed/animal.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative