Copper hydroxide nitrate (Cu2(OH)3(NO3))

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from June 22, 2004 to August 3, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

439-590-3

EC Name:

-

Cas Number:

12158-75-7

Molecular formula:

Cu2H3NO6

IUPAC Name:

copper(2+) hydroxide nitrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

batch n°: 060248
solubility in water: 290 mg/L at 20°C
expiration date of the batch: None. The test substance is stable.
storage conditions of test substance: ambient temperature, no light protection necessary, keep dry
pH of a 1% aqueous suspension: 6.64

Test animals

Species:

mouse

Strain:

other: Crl: NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

hygiene: optimal hygienic conditions
room temperature: average of 21.4°C (continuous control and recording)
relative humidity: average of 61.7% (continuous control and recording)
air exchange: 12 per hour
light: artificial light from 6 a.m. to 6 p.m.
cages: males: single caging in Makrolon cages type II ; females: Makrolon cages type III, low version, 5 animals per cage ; wire mesh lids
bedding material: aspen wood chips, autoclaved ; the bedding material was changed about weekly. Random analyses for contaminants are performed by the supplier ; additionally, a sample of the bedding material is analysed analogously to the feed, once a year.
environmental enrichment: nibbling wood bricks and nesting material, both from the same material and source as the bedding material, were offered to the animals once a week
feed: Altromin standard diet for rats and mice, 1324 forte, germ reduction byv25 kGy 60Co gamma irradiation, ad libitum (producer: Altromin GmbH, D-32761 Lage). Exception: the feed was withdrawn on the day before administration of the test substance at about 5 p.m. and was offered again immediately after dosing. Random samples of the feed are analysed for contaminants by Altromin ; additionally, a feed sample is analysed for contaminants by another independant laboratory once a year.
water: tap water, offered in Makrolon bottles with stainless seel canules, ad libitum. Drinking water is analysed periodically by the "Bundesanstalt für Lebensmitteluntersuchung und -forschung", A-1090 Vienna.
identification: labelling with felt-tipped pen on the tails of the animals and on the cage (colour coding)
acclimatisation: 12 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionised water (+ negative control substance)

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

24 and 48 hours p.a.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
300 mg/Kg body mass
Basis:
nominal in water
dose volume: 10 mL per Kg body mass

Doses / concentrations 2

Remarks:

Doses / Concentrations:
600 mg/Kg body mass
Basis:
nominal in water
dose volume: 10 mL per Kg body mass

Doses / concentrations 3

Remarks:

Doses / Concentrations:
900 mg/Kg body mass
Basis:
nominal in water
dose volume: 10 mL per Kg body mass

No. of animals per sex per dose:

5 ( in dose groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

positive control substance: cyclophosphamide monohydrate (40 mg per Kg body mass)

Examinations

Tissues and cell types examined:

All animals were observed at least once daily for toxic effects of the test substance and for behavioural changes. starting with the day of administration individual records were made. The individual body masses of all animals were recorded a allocation of the animals to the respective group and at administration of the test substance.

Details of tissue and slide preparation:

Animals were killed by cervical dislocation at the appropriate sampling times, i.e. 24 and 48 hours p.a.. bone narrow was obtained from both femurs and was prepared according to the method of W SCHMID. for each animal three smears were prepared. Two of them were stained using a slightly modified Pappenheim method, coded and scored. Decoding was done after the scoring of the last slide.

Evaluation criteria:

Composition of the bone narrow:
For each slide the ratio of nucleated cells to erythrocytes was determined by counting at least 200 cells per slide, i.e. at least 400 cells per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting at least 500 erythrocytes per slide, i.e. at least 1000 erythrocytes per animal.
Micronucleated erythrocytes:
2000 polychromatic erythrocytes per animal were counted (1000 per slide). The number of polychromatic erythrocytes with one or more micronuclei was recorded. When counting erythrocytes for the evaluation of the ratio polychromatic to normochromatic erythrocytes the number of normochromatic erythrocytes with one or more micronuclei was recorded, too. Based on the counted number of normochromatic erythrocytes the percentage of micronucleated normochromatic erythrocytes was calculated. Criteria for the identification of the micronucleus were: the particle had to be round-shaped, of violet or dark blue colour, inside the cell and looking like a compact body when lifting and lowering the objective. Nerly all of the micronuclei had a diameter not smaller than one thenth of the diameter of the enclosing erythrocyte.

Statistics:

Micronucleated cells:
U-test of Wilcoxone, Mann and Witney: for comparison of 2 groups.
H-test of Kruskal and Wallis followed by the test of Nemenyi: for comparison of more than two groups.
Body masses, composition of bone narrow:
t-test: for comparison of two groups.
Analysis of variance followed by the Scheffé test: for comparison of more than two groups.
All statistical analyses were performed seperately for each sex. Additionnally, all parameters investigated were compared between the two sexes of each group.
P = 0.05 was chosen in each test (two-tailed tests).
However for drawing conclusions from the results, also other tham merely statistical considerations, for example biological considerations, are taken into account.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

cytotoxic effect to the bone marrow

Toxicity:

yes

Remarks:

sedation, tremor and signs of reduced well-being like raised fur, arched back or closed eyes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

female

Genotoxicity:

negative

Remarks:

cytotoxic effect to the bone marrow

Toxicity:

yes

Remarks:

sedation, tremor and signs of reduced well-being like raised fur, arched back or closed eyes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mortality, observations in life, body masses:
3/15 high doses males and 3/15 high doses females (spared animals included) died within 24 hours after administration of the test substance. All other animals survived until the scheduled sacrifice. Prematurely died animals were replaced by identically treated spare animals. All teste substance treated animals showed adverse reactions after administration of the test substance. In most animals, the signs lasted until the scheduled sacrifice. Most prominent findings were sedation, tremor, and signs of rduced well-being like raised fur, arched back and closed eyes.
There were no statistically significant differences in mean body masses between the dosed groups and the negative controls, neither at allocation nor at administration of the test substance.
Composition of bone marrow:
The amounts of nucleated cells were statistically significantly lower in high group males 24 and 48 hours after test substance administration, compared to the corresponding negative controls. In females, the difference gained statistical significance only 48 hours p.a. The date were also outside the range of historical negative control data.
the amount of polychromatic erythrocytes were statistically significantly lower in high dose group males 24 hours p.a., in females of the high dose group 24 and 48 hours p.a. and also in mid dose group females 24 hours p.a., compared to the corresponding negative controls. Although all data were within the range of historical negative control data, these findings support the cytotoxic effect of the test substance to the bone marrow.
Micronucleated erythrocytes:
Rate of micronucleated normochromatic erythrocytes: There were no statistically significant differences in the amounts of micronucleated normochromatic erythrocytes between the test substance group animals and the negative controls, neither 24 nor 48 hours after administration.
Rate of micronucleated polychromatic erythrocytes: This is the parameter of major interest. In this test system, a test substance is considered to induce chromosomal damage or damage to the mitotic apparatus, if a statistically significantly increased amount of micronucleated polychromatic erythrocytes compared to the corresponding negative controls is detected and if, additionally, comparison with historical control date shows that this difference is not merely statistically significant.
No relevant differences between the test substance group and the negative control groups were noted in the amounts of micronucleated polychromatic erythrocytes, neither 24 nor 48 hours p.a. and neither for males nor for females. All figures were alsowithin the range of historical negative control data, obtained after oral, intravenous or intraperitoneal administration administration of various negative control substances.
Sex differences:
Some merely statistically significant differences were noted between males and females in the composition of the bone marrow of the negative controls 24 hours p.a. and also in the amounts of micronucleated polychromatic erythrocytes in the low dose group. These differences were not regarded to be of biological importance. No other statistically significant differences between the sexes were noted in the test substance treated groups for any other parameter analysed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Due to the results obtained in this study and under the test conditions described Copper Hydroxide Nitrate did not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes in animals of either sex of the test species at doses of 300, 600 or 900mg Copper Hydroxide Nitrate per Kg body mass and sampling times of 24 and 48 hours p.a. The bioavailability of the test substance was proven by mortality and marked cytotoxicity at the high dose.