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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authority for Biocides.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Principles of method if other than guideline:
The study was not conducted in accordance with any internationally recognised guidelines. However, the methods employed in the study are comparable to OECD Guidelines 486 "Genetic Toxicology: DNA Damage and Repair/Unscheduled DNA Synthesis in Mammalian Cells in vivo".

See Other information on materials and methods.
METHODOLOGY Copper II sulphate pentahydrate was tested using an in vivo/in vitro assay for its ability to cause repairable DNA damage in cultured primary rat hepatocytes. Repair was measured as unscheduled DNA synthesis (UDS) by the uptake of radio-labelled thymidine assayed autoradiographically. The test method follows the techniques described by; Mirsalis, J.C. and Butterworth, B.E., 1980. Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents: An in vivo - in vitro assay for potential mutagens and carcinogens. Carcinogenesis, 1: 621 - 625. Ashby, J., Lefevre P.A., Burlinson, B. and Penman, M.G., 1985. An assessment of the in vivo rat hepatocyte DNA-repair assay. Mutation research, 156: 1-18.

GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Lot/batch number: A668269 350
Description: blue crystalline substance
Purity: 99-100.5 %
Stability: Stable at room temperature
Maximum tolerable dose: <2000 mg/kg

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Source: Charles River UK Ltd, Margate, UK
Sex: Male
Age/weight at study initiation: Test animals were 41-51 days old with a bodyweight range of 189-254 g.
Number of animals per group: 6 animals
Control animals: Yes

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle: purified water
Concentration in vehicle: Not reported
Total volume applied: 10 ml/kg

Positive controls were administered in corn oil or purified water (see Positive controls below).
Duration of treatment / exposure:
Animals were sacrificed either 12-14 hours (experiment 1) or 2-4 hours (experiment 2) after dosing.
Frequency of treatment:
Number of applications: 1
Interval between applications: Not applicable
Post exposure period:
Postexposure period: 12-14 hours for Experiment 1, 2-4 hours for Experiment 2
Doses / concentrations
Remarks:
Doses / Concentrations:
632.5 or 2000 mg/kg
Basis:
actual ingested
Control animals:
yes
Positive control(s):
Purified water was used as the negative control.

7.5 mg/ml 2-Acetamidofluorene (2-AAF) suspended in corn oil was the positive control for the 12-14 hour experiment.

1.0 mg/ml dimethylnitrosamine (DMN) dissolved in purified water was used as the positive control for the 2-4 hour experiment.

Both positive controls were dosed at 10 ml/kg giving achieved doses of 75 mg/kg and 10 mg/kg for the 12-14 and 2-4 hour experiments respectively.

See Table 1 for summary of administered doses.

Examinations

Tissues and cell types examined:
Hepatocytes from the liver.
Details of tissue and slide preparation:
Preparation of hepatocyte cultures:
After either 12-14 hours or 2-4 hours after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from 5 animals in each group and were treated with [3H] thymidine. Six slides were prepared with fixed hepatocytes and of these, 3 were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count, the number of grains present in the nucleus minus the mean number of grains in 3 equivalent areas of cytoplasm, was determined for each of at least 2 of the 3 slides, each animals and dose group.

Number of animals: Cultures from 5 animals were taken.
Number of cells: 150,000 viable cells/ml
Time points: 12-14 hours in Experiment 1, 2-4 hours in Experiment 2.
Evaluation criteria:
The net grain count, number of grains present in the nucleus minus the mean number of grains in 3 equivalent areas of cytoplasm were determined.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clinical signs: Not reported.

Tissue examination: Treatment with copper II sulphate pentahydrate at doses up to 2000 mg/kg yielded group mean net grain counts of less than 0, producing group mean net grain counts over the 2 experiments in the range of -1.0 to -3.2, well below the value of 5 net grain counts required for a positive response. No more than 1.0% of the cells were seen in repair at any dose of test substance.

The data obtained indicate that oral treatment of male rats with 632.5 or 2000 mg/kg copper II sulphate pentahydrate did not result in increased unscheduled DNA synthesis in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing.

The positive control chemicals (2-AAF and DMN) induced increases in the group mean net grain count of 5 or more (12.7 and 17.2 respectively), and 50% or more of the cells (90% and 99.6% respectively) had net grain counts of 5 or more. This result showed that the test system was sensitive to 2 known DNA damaging agents requiring metabolism for their action and that the experiment was valid.

The group mean net grain count for the vehicle-treated animals was less than 0 (-1.3 and -2.2 or Experiments 1 and 2 respectively).

For further information on results, please refer to Table 2a and Table 2b.

Genotoxic: No

Any other information on results incl. tables

Table 2a. Summary of Results.

   Experiment 1: 12-14 hour sacrifice time.

 

Dose (mg/kg)

Net Nuclear Grain Count

Net Grain Count of Cells in Repair

Percent of Cells in Repair (Net Grain Count ≥5)

Mean

SD

Mean

SD

Mean

SD

0

water

-1.3

0.6

0

-

-

-

632.5

Copper II sulphate pentahydrate

-1.3

0.3

10.2

6.4

0.6

0.9

2000

Copper II sulphate pentahydrate

-1.0

0.3

5.5

0.9

1.0

1.0

75 2-AAF

12.7

0.9

13.7

0.8

90.0

4.0

 

 

Table 2b. Summary of Results.

Experiment 2: 2-4 hour sacrifice time.

 

Dose (mg/kg)

Net Nuclear Grain Count

Net Grain Count of Cells in Repair

Percent of Cells in Repair (Net Grain Count ≥5)

Mean

SD

Mean

SD

Mean

SD

0

water

-2.2

0.3

0

-

-

-

632.5

Copper II sulphate pentahydrate

-2.2

0.2

0

-

-

-

2000

Copper II sulphate pentahydrate

-3.2

0.5

0

-

-

-

10 DMN

17.2

2.8

17.3

2.9

99.6

0.9

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Treatment with 632.5 or 2000 mg/kg copper II sulphate pentahydrate did not produce a group mean net grain count greater than -1.0 nor were any more than 1.0% cells found in repair at either dose.
It was concluded that copper II sulphate pentahydrate has no genotoxic activity detectable in this test system under the
experimental conditions employed.
Executive summary:

Materials and Methods

Copper II sulphate pentahydrate was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using an in vivo/in vitro procedure.  Groups of 6 male rats were treated once with copper sulphate at 632.5 or 2000 mg/kg by oral gavage at a dose volume of 10 ml/kg.  For the negative control, a further 6 male rats received purified water as a negative control at the same dose volume.  Positive control animals for the 12-14 hour experiment, 6 male rats were dosed orally with 75 mg/kg 2 -acetamidofluorene, suspended in corn oil.  Dimethylmitrosamine, dissolved in purified water, was the positive control for the 2-4 hour experiment.

Approximately 12-14 hours (experiment 1) or 2-4 hours (experiment 2) after dose administration the animals were sacrificed and the livers perfused with collagenase to provide a primary culture of hepatocytes.  The net grain count, number of grains present in the nucleus, minus the mean number of grains in 3 equivalent areas of cytoplasm were determined.

 

Results and Discussion

Negative control animals gave a group mean net grain of less than 0 with no cells in repair.  Group mean net grain values were increased by both positive controls to more than 5 with more than 50% of cells found to be in repair.  This was consistent with historical control data.

 

Treatment with 632.5 or 2000mg/kg copper sulphate pentahydrate (equivalent to 161 or 509 mg Cu/kg) did not produce a group mean net grain value greater than -1.0 nor were any more than 1.0% cells found in repair at either dose.