Tetracopper hexahydroxide sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential in bacteria of tribasic copper sulphate was assessed by read-across to test results on Copper II sulphate pentahydrate. The test method was designed to meet the requirements of the OECD 471.

No treatment with the read-across substance in any of the test strains, in either the absence or presence of S9, resulted in an increase in revertant numbers that was statistically significant when analysed using Dunnett's test.

By read-across to test results on copper II sulphate pentahydrate, tribasic copper sulphate is considered to be unable to induce mutation Salmonella typhimurium in both the absence and presence of a rat liver metabolic activation system (S9).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 25 March 1994 to 12 April 1994. Report issued: 21 June 1994.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Toxicity and range-finder experiment: 0, 8, 40, 200, 1000 and 5000 µg/plate (+/- S9)
Mutation experiment 1: 0, 1.6, 8, 40, 200 and 1000 µg/plate (+/- S9)
Mutation experiment 2: 0, 50, 100, 200, 400 and 800 µg/plate (+/- S9)

Vehicle / solvent:

Sterile distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For Strain TA98

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA100 and TA1535

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA1537

Positive controls:

yes

Positive control substance:

other: Glutaraldehyde

Remarks:

For strain TA102

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For 1 strain with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In agar using plate incorporation for experiment 1 and preincubation for experiment 2 (preincubation used as the result of the first experiment was negative)

DURATION
- Preincubation period: 1 hour (experiment 2 only)
- Exposure duration: 72 hours

Evaluation criteria:

Acceptance criteria

The assay was considered valid if the following criteria were met:

i) the mean negative control counts fell within the normal ranges for the laboratory
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
iii) no more than 5%, of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria

A test compound was considered to be mutagenic if:

i) the assay was valid as per acceptance criteria above
ii) Dunnett's test gave a significant response (p ≤ 0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible.

Statistics:

Dunnett's test

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Toxicity and dose selection

An initial range-finder experiment was carried out in strain TA100 only, using final concentrations of copper II sulphate pentahydrate at 8, 40, 200, 1000 and 5000 μg/plate. Toxicity was observed following all these treatments of 5000 μg/plate, with further evidence of toxicity (as indicated by a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers) also observed fallowing treatments of 1000 μg/plate.

Due to the toxicity observed in the range-finder experiment, the maximum test dose for treatments of the remaining test strains in Experiment 1 was reduced ta 1000 μg/plate, this being an estimate of the lower limit of toxicity. Evidence of toxicity was again observed following all Experiment 1 treatments of 1000 μg/plate. Some evidence of toxicity was also observed following strain TA102 treatments of 200 μg/plate in the presence of S-9 only.

For Experiment 2 treatments, a maximum test dose of 800 μg/plate was used, this dose being a revised estimate of the lower limit of toxicity.  All Experiment 2 treatments were performed utilising a narrowed dose range, in order to more closely examine those doses of copper II sulphate pentahydrate most likely to exhibit a mutagenic response. Additionally, all Experiment 2 treatments in the presence of s-9 were performed using a pre-incubation step. Evidence of toxicity (and in some cases complete toxicity) was observed fallowing all Experiment 2 treatments of 800 μg/plate. Some treatments in the presence of S9 at lower test doses also produced evidence of toxicity. The higher degree of toxicity observed with Experiment 2 treatments in the presence of S9 was attributed to the use of a pre-incubation step, which allowed an enhanced exposure of the bacteria to the test chemical.

Conclusions:

It was concluded that copper II sulphate pentahydrate was unable to induce mutation in 5 strains of Salmonella typhimurium, when tested at concentrations extending into the toxic range, in both the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Introduction

Copper II sulphate pentahydrate was assessed for its mutagenic potential in bacteria using the following test guidelines:

i)       OECD Test Guideline 471

ii)       EEC Annex V Test B14

Method       

Following a range-finding experiment five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium were treated with copper II sulphate pentahydrate at concentrations ranging between 1.6 and 1000 μg/plate both in the absence and presence of S9 metabolic activation in two separate experiments. Negative ( solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Results

No copper II sulphate pentahydrate treatment of any of the test strains, in either the absence or presence of S9, resulted in an increase in revertant numbers that was statistically significant when analysed using Dunnett's test.

Conclusion

It was concluded that copper II sulphate pentahydrate was unable to induce mutation in five strains of Salmonella typhimurium, when tested at concentrations extending into the toxic range, in both the absence and presence of a rat liver metabolic activation system (S9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus

The potential of tribasic copper sulphate to induce micronuclei in the bone marrow of mice in vivo was assessed by read-across to a study conducted on copper II sulphate pentahydrate.  Although not conducted to the guideline, the test method was comparable to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test).

Mice treated with the read-across source substance exhibited ratios of PCE to NCE that were decreased compared to concurrent vehicle controls when sampled after 24 hours and similar after 48 hours. There were no instances of statistically significant increases in micronucleus frequency for any group receiving the test chemical at either sampling time.

By read-across to copper II sulphate pentahydrate it is concluded that tribasic copper sulphate will not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice.

 

In vivo clastogenic

Treatment with the read-across source substance resulted 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.  

By read-across to copper sulphate pentahydrate, tribasic copper sulphate is expected to be positive for clastogenicity in mice in vivo.

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control

Dose / conc.:

1.1 mg/kg bw (total dose)

Dose / conc.:

1.65 mg/kg bw (total dose)

Dose / conc.:

2 mg/kg bw (total dose)

Dose / conc.:

3.3 mg/kg bw (total dose)

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.

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.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

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

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 07 april 1994 to 17 May 1994. Report issued: 07 July 1994.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

The OECD 474 guideline is not given in the report text, but the study essentially equivalent to it.

Deviations:

not specified

Remarks:

Only one dose tested in the main study, but it is not clear if this is intentional or a deviation from the standard protocol.

GLP compliance:

yes

Type of assay:

other: Mouse Micronucleus

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

An excess number of out-bred CD-1 mice were obtained from Charles River UK Ltd, Margate, UK. They were housed in groups of no more than 3 animals of the same sex in polypropylene cages with wire mesh lids and solid floors containing wood shavings to a depth of approximately 1 cm. Bottled mains (public supply) tap water and food in pellet form were provided ad libitum. Neither of these was known to contain any biological or chemical entity which might interfere with the conduct of the study.

During the period of the study, the room in which the cages were placed was illuminated continuously by fluorescent light for 12 hours out of. Each 24 hour cycle and received at least 15 fresh air changes per hour. All mice were identified by ear tag. Prior to the main study, male and female mice were randomised to groups of 5 using a system of randomly generated numbers. Checks were made on the first day of treatment to ensure group weights differed from the overall mean by no more than 5%.

Route of administration:

oral: gavage

Vehicle:

Purified Water

Details on exposure:

Dosing preparations were made by dissolving Copper II Sulphate Pentahydrate in purified water to give a stock solution from which dose solution were prepared by dilution. Dilutions were made using purified water and the test chemical preparations used within approximately 4 hours of formulation.

Frequency of treatment:

two consecutive days

Dose / conc.:

447 mg/kg bw/day

Remarks:

Main Study

No. of animals per sex per dose:

Vehicle Control: 10 male and 10 females
Treatment group: 15 male and 15 female (included additional 5 mice to be used in the event of deaths among simiilarly dosed animals
Positive Control: 5 male and 5 female

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 80 mg/kg bw/day

Evaluation criteria:

Acceptance Criteria
The assay was to be considered valid if the following criteria were met:

1) The heterogeneity test provided evidence of acceptable variability between animals within a group
2) The incidence of micronucleated PCE in vehicle control groups fell within or close to the historical vehicle control range.
3) At least 8 animals (males plus females) out of each group at each kill time were available for analysis.
4) The positive control chemical ( CPA) induced statistically significant increases in the frequency of micronucleated PCE.


Evaluation Criteria
The test chemical is considered to be clearly positive if:

1) A statistically significant increase in the frequency of micronucleated PCE occurred for at least one kill time.
2) The frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
3) Corroborating evidence was obtained, for example, increased but statistically insignificant frequencies of micronucleated PCE at the other kill time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Selection of doses for main study

The range-finding study was conducted using groups of 3 males and 3 females. The pattern of mortality 2 days after the second dose is given in the table below:

	Observed Deaths
Dose (mg/kg bw)	Males	Females
142.8	0	0
219.7	0	0
338	0	0
520	1	1
800	2	0
1231	3	3
1893	3	3
2000	3	3

The calculated LD50 was approximately 745 mg/kg. A dose equivalent to 50-80% of the LD50 is considered acceptable as a maximum dose level so 447 mg/kg (60%) was chosen as the upper dose for the micronucleus assay.

Analysis of data

Mice treated with Copper II Sulphate Pentahydrate exhibited PCE/NCE ratios which were decreased compared to concurrent vehicle controls at the 24 hour sampling time. This is indicative of cellular toxicity and evidence of Copper II Sulphate Pentahydrate penetration into the bone marrow. Mice sampled at 48 hours after treatment with Copper II sulphate Pentahydrate exhibited PCE/NCE ratios which were similar to those in vehicle controls. The numbers of micronucleated PCE seen at both sampling times were similar to those seen in controls and were not significantly different by X² analysis.

Conclusions:

It was concluded that Copper II Sulphate Pentahydrate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated at 447 mg/kg, a dose at which limited mortality was observed.

Executive summary:

Introduction

The study was conducted to assess the potential of Copper II Sulphate Pentahydrate to induce micronuclei in the bone marrow of mice in vivo.  Although not conducted to the guideline, the test method was comparable to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test).

Method

The choice of dose level was based on an initial toxicity range-finder study in which Copper II Sulphate Pentahydrate, made up in purified water, was administered to mice by oral gavage.

For the micronucleus test, Copper II Sulphate Pentahydrate was administered once daily, by oral gavage, for 2 consecutive days at 447 mg/kg (113. 76 mg Cu II/kg, 60% of the LD50) to groups of 5 male and 5 female mice. The mice were killed 24 or 48 hours after the second administration.

Several animals receiving Copper II Sulphate Pentahydrate died prior to the scheduled sampling times indicating that it would not have been possible to administer the test chemical at an appreciably higher dose.

The negative (vehicle) control in the study was purified water, also administered by oral gavage on 2 consecutive days. Groups of 5 male and 5 female mice treated with this were killed and sampled after 24 or 48 hours. cyclophosphamide (CPA), the positive control, was dissolved in purified water and administered by oral gavage as a single dose at 80 mg/kg to groups of 5 male and 5 female mice which were killed after 24 hours.

Results

All positive control animals exhibited significantly increased numbers of micronucleated polychromatic erythrocytes (PCE).  Negative (vehicle) control mice exhibited normal ratios of PCE to NCE (norrnochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control ranges.

Mice treated with Copper II Sulphate Pentahydrate exhibited ratios of PCE to NCE that were decreased compared to concurrent vehicle controls when sampled after 24 hours, which was taken as evidence of Copper II Sulphate Pentahydrate absorption into the bone marrow. The PCE/NCE ratios seen in animals sampled at 48 hours were similar to those seen in vehicle controls. Copper II Sulphate Pentahydrate exhibited frequencies of micronucleated PCE which were similar to vehicle controls at all sampling times. There were no instances of statistically significant increases in micronucleus frequency for any group receiving the test chemical at either sampling time.

Conclusion

It is concluded that Copper II Sulphate Pentahydrate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated at 447 mg/kg/day (113.76  mg Cu II/kg/day), a dose at which limited mortality was observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

The results obtained on the read-across substance, copper II sulphate pentahydrate show no concern for humans in regard to the mutagenic effects. The same mutagenic response is expected in the structurally similar substance, copper tribasic sulphate. No classification is therefore required.