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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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 Authorities for Biocides and Existing Substance Regulations.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Cited as Directive 2000/32/EC, B.12
Deviations:
yes
Principles of method if other than guideline:
Deviations: Following test termination, test animals were sacrificed and bone marrow extracted from both femurs of each test animal. However with one test animal (2338) only one femur was aspirated.
This was not considered to have affected the outcome of the study.
GLP compliance:
yes
Type of assay:
micronucleus assay

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: 338 mg/kg

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River, UK Ltd, Margate, UK

Age/weight at test initiation: Ages ranged from 35-42 days for both males and females. Bodyweight ranged from 24-30 g and 21-26 g for males and females respectively.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Vehicle used: Purified water.
Total volume applied: 20ml/kg.
Frequency of treatment:
Two applications with an interval of 24 hours.
Post exposure period:
Test animals were sacrificed at either 24 or 48 hours following the second dose administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
447 mg/kg
Basis:

No. of animals per sex per dose:
Number of animals per group: 15 males and 15 females were treated with the test substance (this includes and additional 5 mice per sex to be used in the event of deaths among similarly dosed animals), 10 males and 10 females were treated with the negative control and 5 males and 5 females were treated with the positive control.
Control animals:
yes
Positive control(s):
Controls: Cyclophosphamide (CPA) was dissolved in purified water at 4 mg/ml to serve as a positive control, and administered at 80 mg/kg with a dose volume of 20 ml/kg. The positive control was administered as a single dose. The negative control was purified water administered twice at the same sampling points as the test substance.

Examinations

Tissues and cell types examined:
Erythrocytes in bone marrow.
Evaluation criteria:
Parameters analysed: polychromatic/normochromatic erythrocytes ratio.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
See additional information on results.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clinical signs: Not reported.

Tissue examination: Mice treated with copper II sulphate pentahydrate exhibited polychromatic/normochromatic erythrocytes (PCE/NCE) ratios which were decreased compared to concurrent vehicle controls at 24 hour sampling point. This is indicative of cellular toxicity and evidence of the test substance penetration into the bone marrow. Mice sampled at 48 hours after being treated with copper II sulphate pentahydrate exhibited ratios which were similar to those in the vehicle controls. The number of micronucleated PCE seen at both sampling times were similar to those seen in the controls and were not significantly different by x2 analysis.

The positive control induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

See Table 1.

Any other information on results incl. tables

Table 1. Summary of Group Mean Data.

 

Treatment group (mg/kg x2)

Sampling point (hours)

Sex

Mean ratio PCE/NCE

Group mean frequency of micronucleated PCE (per 1000)

Per sex

Per treatment group

Vehicle control (purified water)

24

Male

1.07

0.40

0.35

Female

1.20

0.30

48

Male

1.44

0.38

0.33

Female

0.83

0.30

447 (copper sulphate pentahydrate)

24

Male

0.70

0.60

0.50

Female

0.84

0.40

48

Male

1.12

0.50

0.45

Female

1.32

0.40

Positive control (CPA – single dose only)

24

Male

0.52

26.87

28.07

Female

0.48

29.27

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that copper II sulphate pentahydrate did not induce micronuclei in the polychromatic erthrocytes of the bone marrow of mice treated at 447 mg/kg/day, a dose at which limited mortality was observed. The test substance was therefore not genotoxic.
Executive summary:

Copper II sulphate pentahydrate was assayed in vivo in a mouse bone marrow micronucleus test at a single dose level of 447 mg/kg (113.76 mg Cu/kg) for two consecutive days to groups of 5 male and 5 female mice sacrificed 24 or 48 hours after the second administration. Both negative (purified water) and positive controls (cyclophosphamide) were included in the study. The study was conducted according to EEC Annex V test B12 guidelines and in compliance with GLP.

Mice treated with copper II sulphate pentahydrate exhibited frequencies of micronucleated polychromatic erthrocytes 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 point.