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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

March 4, 2004 to November 14, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Copper sulfate

IUPAC Name:

Copper sulfate

Constituent 2

Reference substance name:

7758-99-8

Cas Number:

7758-99-8

IUPAC Name:

7758-99-8

Test material form:

solid: crystalline

Details on test material:

The test substance, a blue crystalline solid of known purity and composition, was purchased from Sigma-Aldrich and assigned Haskell Number 26309. A purity value of 100% was used in the calculation of the amount of test substance to add to the diet.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal rooms were maintained at an acceptable temperature of 18-26ºC (targeted at 22º-24ºC) and maintained at an acceptable relative humidity of 30%-70% (targeted at 40%- 60%). Occasional excursions outside the accepted ranges were minor and did not affect the study. Animal rooms were artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Diet Preparation:
The test substance was pulverized using a mortar and pestle, under a nitrogen blanket. A small amount of rodent diet was then added to the test substance. This test substance/rodent diet premix was added to the remaining rodent diet in the mixer and mixed for 6 minutes. The rodent diet used for the control group was also mixed for 6 minutes in the diet mixer. Neither the amount nor nature of the contaminants in the feed was expected to affect the integrity or validity of the study. Diets were prepared weekly and refrigerated until used. Details of diet preparation are documented in study records.

Administration:
The test substance was administered orally as it is a potential route for human exposure. During the test period, rats in each group was fed a diet of PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 that contained the test substance at the concentrations specified in the
experimental design.

Details on mating procedure:

Following at least 70 days of diet administration (premating), the P1 and F1 generation males and females were co-housed within their respective
treatment groups to produce F1 and F2 litters, respectively. Dams were allowed to deliver and rear their offspring until weaning (postpartum day 21). At weaning, 30 F1 rats/sex/group were randomly selected to comprise the F1 generation and were given the same dietary concentration level as their respective P1 generation sires and dams. F1 and F2 litters were culled to 4 pups/sex/litter (litter size permitting) on postnatal day 4; all remaining pups were discarded without further evaluation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration verification data indicate that the test substance was present at the targeted levels during the study. Based on this information, it can be concluded that the animals received the targeted dietary concentrations of test material during the study. The mean concentration of
copper in the control diet was 13.7 ppm. The range of targeted concentrations added to the diet was 25 - 382 ppm (equivalent to 100 – 1500 ppm copper sulfate).

Duration of treatment / exposure:

Approximate age at start of dosing:
P1 generation: 56 days
F1 generation: 21

Approximate number of study days before mating:
P1 generation: 70 days (at least)
F1 generation: 70 days (at least)

Duration of dosing:
until sacrifice

Frequency of treatment:

daily

Details on study schedule:

Sacrifice schedule:
Adult males: P1 = test days 109-113 and F1 = test day 119
Pregnant females: P1, F1 = on day of weaning litters (day 21 postpartum)
Non-pregnant females: P1, F1 = approximately day 28 after the end of cohabitation
Culled pups: F1, F2 = day 4 postpartum
Weanlings: F1, F2 = on day of weaning (except F1 rats selected as parental rats)

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 10, 500, 1000, 1500 ppm
Basis:
other: in feed

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

Clinical observations, body weight, and food consumption were determined weekly thoughout the study.

Oestrous cyclicity (parental animals):

Estrous cycle parameters (percent days in diestrus, proestrus and estrus) and estrous cycle length were evaluated for 3 weeks prior to cohabitation in P1 and F1 rats.

Sperm parameters (parental animals):

Sperm motility, morphology and concentration in the cauda epididymis and spermatid concentration in the testis were determined.

Litter observations:

Litter examinations including number of live and dead, individual pup weights, clinical observations) were determined at birth, on day 4 and weekly during the lactation period.

Postmortem examinations (parental animals):

All P1 and F1 generation rats that were not found dead, were sacrificed by carbon dioxide euthanasia and exsanguination. All P1 and F1 rats received a gross pathological examination.
The uteri of all cohabited females were examined for the presence and number of implantation sites.
Blood (approximately 2 mL) was collected from the caudal vena cava from the first 10 rats (the first 10 consecutive animal numbers in each group) surviving to scheduled sacrifice in each of Copper Sulfate Pentahydrate: Multigeneration Reproduction Study in Rats DuPont-14226- 28 - the P1 and F1 male and female groups. Scheduled sacrifice for these rats was in the morning (to optimize copper content in the blood samples). Blood samples were placed into EDTA tubes, processed to plasma, and frozen at approximately -80°C.
Tissues were collected from all P1 and F1 adult animals and preserved in appropriate fixative for possible future histopathological examination.

Postmortem examinations (offspring):

Pups were euthanized by decapitation (for pups up to lactation day 4) or by carbon dioxide euthanasia and exsanguination. Pups that died (found dead, sacrificed in extremis, or accidentally killed) during the lactation period underwent a gross pathological evaluation and the carcass was preserved in an appropriate fixative.
At culling on lactation day 4, twelve randomly selected pups (6 male and 6 female) per dose group had samples of liver and brain collected, frozen in liquid nitrogen and stored at approximately -80°C.

Statistics:

Parameters : body weight, body weight gain, food consumption, food efficiency, gestation length, implantation site numbers, implantation efficiency, mean number of pups per litter, percent born alive, 0-4 day viability, viability index, lactation index, precoital interval, vaginal patency, preputial separation, estrous cycle parameters, sperm parameters, ovarian follicle counts, organ weight
Preliminary test: Levene’s test for homogeneity and Shapiro-Wilk test for normality
If preliminary test is not significant: One-way analysis of variance and Dunnett's test
If preliminary test is significant: Kruskal-Wallis test and Dunn's test

Parameters: incidence of clinical observations, mating index, fertility index, gestation index, litter survival
Preliminary test: none
If preliminary test is not significant or significant: Cochran-Armitage test for trend

Parameters: sex ratio (covariate: litter size), mean pup weights (covariates: litter size)
Preliminary test: Levene’s test for homogeneity and Shapiro-Wilk test for normality
If preliminary test is not significant: Analysis of covariance and Dunnett-Hsu
If preliminary test is significant: Non-parametric analysis of covariance

Reproductive indices:

Calculated for P1 and F1 parental animals: mating index(%), fertility index (%), gestation index (%), implantation efficiency (%), pups born alive (%), viability index (%), lactatio index (%), litter survival (%).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased spleen weight in P1 adult females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased spleen weight in P1 adult females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest concentration tested

Remarks on result:

other: Generation: P1, F1, F2 (migrated information)

2

Dose descriptor:

NOEL

Remarks:

lactation

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decreased spleen weight in P1 adult females, and F1 and F2 male and female weanlings.

Remarks on result:

other: Generation: P1 adult females, F1 and F2 male and female weanlings (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased spleen wieight in F1 and F2 male and female weanlings

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

decreased spleen wieight in F1 and F2 male and female weanlings

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL) for reproductive toxicity was 1500 ppm of Copper sulfate pentahydrate, the highest concentration tested. The NOEL for P1 and F1 rats and F1 and F2 offspring during lactation was 1000 ppm of Copper sulfate pentahydrate, based on reduced spleen weight in P1 adult females, and F1 and F2 male and female weanlings at 1500 ppm of Copper sulfate pentahydrate. The dietary concentration of 1000 ppm of Copper sulfate pentahydrate was equivalent to mean daily intakes of Copper of 15.2-23.5 mg/kg body weight/day for male rats during premating and 17.0-26.7 mg/kg body weight/day for female rats during premating and gestation.