Tetracopper hexahydroxide sulphate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Copper sulphate

EC Number:

231-847-6

EC Name:

Copper sulphate

Cas Number:

7758-99-8

Molecular formula:

CuSO4.5H2O

Test material form:

solid: crystalline

Method

Target gene:

Histidine

Species / strain

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For Strain TA98

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For strains TA100 and TA1535

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA1537

Controls 5

Positive controls:

yes

Positive control substance:

other: Glutaraldehyde

Remarks:

For strain TA102

Controls 6

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).