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

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The animal studies of cupric sulfate were performed in compliance with U.S. Food and Drug Administration Good Laboratory Practices regulations (21 CFR 58). The Quality Assurance Unit of Battelle performed audits and inspections of protocols, procedures, data, and reports throughout the course of the studies. Free ionic copper is considered the active ingredient in inorganic copper salts, and is believed to be responsible for adverse effects. The bioavailablity of the copper ion at target sites determines the severity of effects. Read-across from copper sulfate is a conservative approach based on differences in water solubility. Copper sulfate is more water soluble than copper hydroxide nitrate, and therefore the bioavailablity of the copper ion from copper hydroxide nitrate is less that from copper sulfate.

Qualifier:

according to guideline

Guideline:

other: not specified

Principles of method if other than guideline:

Two-week drinking water studies and Two-week and 13-week dosed feed studies of cupric sulfate toxicity were carried out with male and female F344/N rats and B6C3F mice. In the 13-week studies, gross and histopathologic examinations and sperm morphology and vaginal cytology evaluations were performed on rats and mice, anc clinical pathology anaylses were conducted for rats.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Fischer 344/N rats and B63F mice

Route of administration:

oral: feed

Vehicle:

other: meal flour

Details on oral exposure:

Dosed feed was available ad libitum for 92 days. Copper sulfate at concentrations of 0, 500, 1000, 2000, 4000, and 8000 ppm was administered to 10 rats/sex/exposure group for the full 92 days and 10 rats/sex/exposure group for special studies.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses indicated that all dose formulations were within ± 10% of theoretical concentrations.

Duration of treatment / exposure:

13 weeks

No. of animals per sex per dose:

Dosed feed was available ad libitum for 92 days. Copper sulfate at concentrations of 0, 500, 1000, 2000, 4000, and 8000 ppm was administered to 10 rats/sex/exposure group for the full 92 days and 10 rats/sex/exposure group for special studies.

Details on study design:

Cupric sulfate concentrations used in the 2-week dosed water and dosed feed studies were selected based on LD50 values reported in the literature, and the concentrations used in the 13-week studies were selected based on the results of the 2-week studies. For the 2-week studies, groups of 5 rats and 5 mice of each sex were administered cupric sulfate at concentrations of 0,300, 1000,3000, and 10,000 ppm in drinking water or 0, 1000, 2000, 4000, 8000, and 16,000 ppm in dosed feed for 15 days. In the 13-week dosed feed studies, dosed feed was available ad libitum for 92 days. Cupric sulfate at concentrations of 0, 500, 1000, 2000, 4000, and 8000 ppm was administered to 10 rats/sex/exposure group for the base study groups (full 92 days of dosing) and to an additional 10 rats/sex/exposure group for special studies (intermediate time points for clinical pathology determinations). Groups of 10 mice/sex/exposure group received cupric sulfate at 0, 1000, 2000, 4000, 8000, and 16,000 ppm for 92 days. A complete necropsy was performed on all early death animals and at the termination of each study on all treated and control animals. Body weights and the weights of the liver, thymus, right kidney, right testis, heart, lungs, and brain were determined. Organs and tissues were examined for gross lesions and fixed in 10% neutral buffered formalin. A standard battery of 34 organs and tissues (NTP, 1992) were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Complete histopathologic examinations were carried out on all control animals, all early death animals, all animals in the highest dose group with at least 60% survivors, and all animals in higher dose groups. Organs identified as target organs (liver, kidney, and forestomach) were examined to a no-effect level in lower exposure groups.

Observations and examinations performed and frequency:

A complete necropsy was performed on all early death animals and at the termination of each study on all treated and control animals. Body weights and the weights of the liver, thymus, right kidney, right testis, heart, lungs, and brain were determined. Organs and tissues were examined for gross lesions and fixed in 10% neutral buffered formalin. A standard battery of 34 organs and tissues (NTP, 1992) were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Complete histopathologic examinations were carried out on all control animals, all early death animals, all animals in the highest dose group with at least 60% survivors, and all animals in higher dose groups. Organs identified as target organs (liver, kidney, and forestomach) were examined to a no-effect level in lower exposure groups.

Statistics:

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response (Dunnett, Dunn). The outlier test of Dixon and Massey (1951) was employed to detect extreme values. Because the vaginal cytology data are proportions, an arcsine transformation was used to bring the data into closer conformance with normality assumptions. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for the simultaneous equality of measurements across dose levels.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results:

Administration of cupric sulfate to rats in feed or drinking water resulted in significant gastric changes and hepatic and renal damage. The primary lesion in rats was an increase in the size and number of proteinaceous droplets in the epithelial cytoplasm and lumen of the proximal convoluted tubule. For rats in the 13-week study, the no observed-adverse-effect level (NOAEL) for evidence of histologic injury to the kidney was 1000 ppm for males and 500 ppm for females, while the NOAEL for liver inflammation was 1000 ppm for males and 2000 ppm for females. Hyperplasia with hyperkeratosis of the epithelium on the limiting ridge separating the forestomach from the glandular stomach was also seen in rats of each sex, and the NOAEL for this change was 1000 ppm cupric sulfate in the feed. Additionally, clinical pathology alterations noted in the 13-week study, along with histologic changes in bone marrow noted in the 2-week feed study, were indicative of a microcytic anemia with a compensatory bone marrow response. Mice
appeared to be much more resistant to the toxic effects of cupric sulfate than rats. The primary target tissue in mice was the epithelium of the limiting ridge of the forestomach. The NOAEL for the hyperplasia and hyperkeratosis seen at this site in mice was 2000 ppm cupric sulfate in the feed. The NOAEL in male rats for the 13-week study was 64 mg/kg/day as cupric sulfate.

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Hyperplasia and hyperkeratosis of the forestomach mucosa. Damage to liver, kidneys, and the hematopoietic system.

Critical effects observed:

not specified

Conclusions:

Administration of cupric sulfate to rats in feed or drinking water resulted in significant gastric changes and hepatic and renal damage. The primary lesion in rats was an increase in the size and number of proteinaceous droplets in the epithelial cytoplasm and lumen of the proximal convoluted tubule. For rats in the 13-week study, the no observed-adverse-effect level (NOAEL) for evidence of histologic injury to the kidney was 1000 ppm for males and 500 ppm for females, while the NOAEL for liver inflammation was 1000 ppm for males and 2000 ppm for females. Hyperplasia with hyperkeratosis of the epithelium on the limiting ridge separating the forestomach from the glandular stomach was also seen in rats of each sex, and the NOAEL for this change was 1000 ppm cupric sulfate in the feed. Additionally, clinical pathology alterations noted in the 13-week study, along with histologic changes in bone marrow noted in the 2-week feed study, were indicative of a microcytic anemia with a compensatory bone marrow response. Mice
appeared to be much more resistant to the toxic effects of cupric sulfate than rats. The primary target tissue in mice was the epithelium of the limiting ridge of the forestomach. The NOAEL for the hyperplasia and hyperkeratosis seen at this site in mice was 2000 ppm cupric sulfate in the feed. The NOAEL in male rats for the 13-week study was 64 mg/kg/day as cupric sulfate.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

16 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

Free ionic copper is considered the active ingredient in inorganic copper salts, and is believed to be responsible for adverse effects. The bioavailablity of the copper ion at target sites determines the severity of effects. Read-across from copper sulfate is a conservative approach based on differences in water solubility. Copper sulfate is more water soluble than copper hydroxide nitrate, and therefore the bioavailablity of the copper ion from copper hydroxide nitrate is less that from copper sulfate.

Administration of cupric sulfate to rats in feed or drinking water resulted in significant gastric changes and hepatic and renal damage. The primary lesion in rats was an increase in the size and number of proteinaceous droplets in the epithelial cytoplasm and lumen of the proximal convoluted tubule. For rats in the 13-week study, the no observed-adverse-effect level (NOAEL) for evidence of histologic injury to the kidney was 1000 ppm for males and 500 ppm for females, while the NOAEL for liver inflammation was 1000 ppm for males and 2000 ppm for females. Hyperplasia with hyperkeratosis of the epithelium on the limiting ridge separating the forestomach from the glandular stomach was also seen in rats of each sex, and the NOAEL for this change was 1000 ppm cupric sulfate in the feed. Additionally, clinical pathology alterations noted in the 13-week study, along with histologic changes in bone marrow noted in the 2-week feed study, were indicative of a microcytic anemia with a compensatory bone marrow response. Mice

appeared to be much more resistant to the toxic effects of cupric sulfate than rats. The primary target tissue in mice was the epithelium of the limiting ridge of the forestomach. The NOAEL for the hyperplasia and hyperkeratosis seen at this site in mice was 2000 ppm cupric sulfate in the feed. The NOAEL in male rats for the 13-week study was 64 mg/kg/day as cupric sulfate.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: other

Justification for classification or non-classification

According to the EC Regulation No. 1272/2008 and subsequent regulations, Basic Copper Nitrate is not to be classified for the 'Repeated dose toxicity' endpoints.