Tetracopper hexahydroxide sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Test material form:

solid

Details on test material:

- Source: Glaxo Laboratories India Ltd, India.

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Departmental Animal House, University of Calcutta
- Age at study initiation: 8- 10 weeks
- Weight at study initiation: 20 - 25g
- Assigned to test groups randomly: not stated
- Fasting period before study: No
- Housing:Polycarbonate cages at no more than 8 animals per cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 15%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Isotonic saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance was prepared in isotonic saline prior to dosing.

Duration of treatment / exposure:

A single dose of test substance was administered by intraperitoneal injection and animals were killed at 6, 12 or 24 hours by cervical dislocation.

Frequency of treatment:

One treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

1.1 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

1.65 mg/kg bw (total dose)

Doses / concentrations 4

Dose / conc.:

2 mg/kg bw (total dose)

Doses / concentrations 5

Dose / conc.:

3.3 mg/kg bw (total dose)

Doses / concentrations 6

Dose / conc.:

6.6 mg/kg bw (total dose)

No. of animals per sex per dose:

Six males per dose at each time point.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C at 1.5 mg/kg bw administered by intraperitoneal injection. The animals were killed at 6 hours after dosing.

Examinations

Tissues and cell types examined:

Bone marrow of both femurs from each animal.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Each bone marrow sample was washed twice in fixative and slides were prepared by flame drying, coded and stained in diluted Giemsa.

METHOD OF ANALYSIS:
All the slides were observed under oil immersion lens. 50 metaphase plates from each of the 6 animals per dose were scored, the selection being based on uniform staining quality, lack of overlapping chromosomes and chromosome number (40 ± 2). Individual types of aberrations (i.e., chromatid vs. chromosome gaps, breaks and rearrangements) were recorded separately. Data were evaluated as the per cent aberrant metaphase cells (excluding gaps) and as the number of aberrations per cell (excluding gaps). All aberrations were considered equal regardless of the probable number of breakage events involved.

Statistics:

A 1-tailed trend test was used to determine if a treatment-related increase occurred. A 2-way ANOVA test followed by Duncan's multiple range test was carried out to observe the significant differences, if any, amongst the different concentrations and sampling times on the clastogenicity.

The level of significance was established at P = 0.05 for all analyses.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

The following table summarises the results of the test:

Chromosomal Aberrations

Exposure (hours)	Mitomycin C 1.5 mg/kg bw    (Positive Control)	Test Substance Dose Level (mg/kg bw)
		0	1.1	1.65	2	3.3	6.6
Chromosome Aberrations (excluding gaps)
6	0.077	0.017	0.053	0.06	0.073	0.067	0.1
12	---	0.017	0.023	0.040	0.037	0.05	0.087
24	---	0.01	0.037	0.047	0.047	0.040	0.05
% damaged cells with at least 1 aberration
6	7.667	1.670	5.330	6.000	7.330	6,670	10.00
12	---	1.670	2.330	4.000	3.670	5.000	8.670
24	---	3.670	4.670	4.670	4.670	4.000	5.000

 Note: table is reproduced from CLH Report for Tribasic Copper Sulphate

Applicant's summary and conclusion

Conclusions:

The test substance gave a positive response and was considered to be a moderately clastogenic agent in mice in vivo.

Executive summary:

Introduction

The in vivo clastogenic effect of the test substance on the bone marrow chromosomes of mice was investigate using a method similar to OECD 475 Mammalian Bone Marrow Chromosome Aberration Test.

 

Method

The test substance dissolved in isotonic saline, was administered by intraperitoneal injection to Swiss albino male mice at dose levels of 1.1, 1.65, 2.0, 3.3 and 6.6 mg/kg. Groups of six mice were killed at 6, 12 and 24 h after treatment for each dose. Prior to sacrifice the mice were injected with 4 mg/kg colchicine to inhibit mitosis. A positive control group of mice was treated with 1.5 mg/kg mitomycin C (a positive control article) and then animals killed after 6 h. Bone marrow smears were prepared by standard methods and 50 metaphases from each of the six animals from each group were scored for aberrations, excluding gaps.

 

Results

Treatment with the test substance at results in chromosome aberrations at all concentrations with the degree of clastogenicity directly related to concentrations use and indirectly to period of exposure. The clastogenic effect was maximal at six hours after treatment compared to 12 and 24 hours indicating an early onset of clastogenesis.  

Conclusion

The test substance gave a positive response and was considered to be a moderately clastogenic agent in mice in vivo.