Basic copper zinc nitrate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-04 to 2004-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001-01-22

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

7758-99-8

Molecular formula:

CuSO4 · 5H2O

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): copper sulfate pentahydrate
- State of aggregation: blue crystals

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was pulverized using a mortar and pestle, under a nitrogen blanket.

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Details on species / strain selection:

The rat was selected for this study as it is a preferred species for reproductive toxicity testing as recommended by regulatory guidelines. The Crl:CD®(SD)BR strain was chosen because extensive background data are available from the literature, the supplier, and previous studies with other compounds at the laboratory. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: P0 generation: approx. 56 days; F1 generation: approx. 21 days
- Weight at study initiation (P0 generation): males: approx. 262 - 332 g; females: approx. 166 - 231 g

- Housing:
males: housed individually during non-mating periods in stainless steel, wire-mesh cages
pretest and premating period: all rats were housed individually in stainless steel, wire-mesh cages
cohabitation period: all rats were housed as breeding pairs in stainless steel, wire-mesh cages; at the end of the cohabitation period, females without evidence of copulation were housed individually in polycarbonate pans.
gestation period (females with evidence of copulation; days 0 - 20): dams were housed individually in stainless steel, wire-mesh cages
gestation period (females with evidence of copulation; day 20 - delivery): females were housed individually in polycarbonate pans with bedding
lactation period: dams were housed with their litters in polycarbonate pans with bedding.

- Diet (ad libitum): PMI® Nutrition International, Certified Rodent LabDiet® 5002 meal (copper content: 13.7 ppm)
- Water (ad libitum): tap water (copper content (ICP): 0.019 ppm)
- Quarantine period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 26ºC (targeted at 22º-24ºC)
- Relative humidity: 30 % - 70 % (targeted at 40 %- 60 %)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Purity value of 100% was used in the calculation of the amount of test substance to add to the diet.
- Preparation of diet: a small amount of rodent diet was added to the pulverized 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.
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: refrigerated

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female (rats were nonsiblings)
- Length of cohabitation: cohoused until evidence of copulation was observed or until 2 weeks had elapsed. The cohabitation period ended in the morning of day 15 of cohabitation.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): individual cage housing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration verification diet samples containing copper sulfate at concentrations of 100, 500, 1000 and 1500 ppm (equivalent to 100, 500, 1000, and 1500 mg/kg) were submitted for analysis at the initiation of this study. Also, diet samples for the same levels were submitted and stored to establish stability of test substance in diet (0, 7, and 14 days at room temperature and 14, and 21 days refrigerated), then analyzed. Diet samples at all levels were submitted for concentration verification analysis on test day 111 and test day 237. In addition, 0 ppm diet samples were included with each submittal.
The concentrations of copper sulfate in separate diet samples were determined by measurement with Inductively Coupled Plasma - Atomic Emission Spectroscopy (ICP-AES).

Results:
The homogeneity data show that the mixing procedure used in this study distributed copper sulfate uniformly throughout the diet. The measured values for the individual homogeneity samples were within ± 12.4% of nominal with the mean measured values 90.1%, 93.8%, 87.6%, and 90.7% of nominal and C.V.’s of 4%, 9%, 3%, and 10% for the respective dietary levels (100, 500, 1000, and 1500 ppm).
The measured values for the stability samples were from 82.5% to 111.0% of nominal for all dietary levels and all storage conditions (up to 14 days at room temperature and 21 days refrigerated) showing that copper sulfate is stable when mixed in the diet and stored at room temperature for up to 14 days or refrigerated for up to 21 days.
The measured values for the concentration verification samples (test days 111 and 237) ranged from 82.0 to 107.0% of nominal. The mean % nominal for all samples was 96.7% ±
9.3 with a CV = 9.6%. The results were within expected variability of the analytical method.
The copper as analyzed in the control diet agreed with the specified amount of copper that was reported by supplier to be in the diet. All reported results for the study diet samples
were corrected for the analyzed value of the copper in the control.

Duration of treatment / exposure:

males (P0): 109 - 113 days
males (P1): 119 days
pregnant females: approx. 120 days
nonpregnant females: approx 112 days

Frequency of treatment:

ad libitum

Details on study schedule:

NOTE: F1 animals selected for mating were named P1 in the result section, since they are considered to be the second parental generation.
- F1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age (one rat/sex/litter when possible).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg diet

Doses / concentrations 2

Dose / conc.:

500 mg/kg diet

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg diet

Doses / concentrations 4

Dose / conc.:

1 500 mg/kg diet

No. of animals per sex per dose:

30 males / 30 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: in a 90-day feeding study (Hebert etal., 1993)* groups of 10 rats per sex were fed diets containing 0, 500, 1000, 2000, 4000, or 8000 ppm of cupric sulfate (equivalent to 0, 500, 1000, 2000, 4000, or 8000 mg/kg). There was no test substance-related mortality or clinical signs of toxicity in the study. Mean final body weight and overall body weight gain were lower than control at 4000 ppm (males) and 8000 ppm (males and females). Overall food consumption was lower than control at 8000 ppm (males and females). There were microscopic lesions in the fore-stomach and liver at ≥ 2000 ppm and lesions in the kidney at ≥ 1000 ppm. There were test substance-related changes in hematologic, clinical chemistry and urinalysis parameters that were, for the most part, restricted to the 4000 and 8000 ppm groups.
Dietary levels of 0, 100, 500, 1000, and 1500 ppm (equivalent tp 0, 100, 500, 1000 and 1500 mg/kg) were selected for the current study. The dietary level of 1000 ppm was expected to produce no or minimal toxic effects. The 1500 ppm level was expected to produce some systemic toxicity but no mortality. The 500 ppm level was expected to be the no-observed-effect level (NOEL).

*Reference:
- Hebert, C.D., Elwell, M.R., Travlos, G.S., Fitz, C.J., Bucher, J.R. (November, 1993). Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice. Fundam Appl Toxicol. 21(4):461-75.

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

NOTE: F1 animals selected for mating were named P1 in the result section, since they are considered to be the second parental generation.

CAGE SIDE OBSERVATIONS: Yes (P0 and F1 parental generation)
- Time schedule: at least once daily throughout the study; moribund rats were sacrificed

DETAILED CLINICAL OBSERVATIONS: Yes (P0 and F1 parental generation)
- Time schedule: at least once weekly throughout the premating feeding, gestation, and lactation period

BODY WEIGHT: Yes (P0 and F1 parental generation)
- Time schedule for examinations:
premating period (all rats): once a week; all F1 rats evaluated for developmental landmarks (vaginal patency, preputial separation) were weighed on the day of achievement
gestation and lactation periods (dams): days 0, 7, 14, and 21 of each period
gestation and lactation periods (females without evidence of copulation, those that copulated and did not deliver a litter, and males): weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (P0 and F1 parental generation):
Premating and cohabitation period:
- individual food consumption was determined weekly for all P0 and F1 rats throughout the period, ending on test day 70.
- food consumption was not measured during cohabitation for males and females or after cohabitation for males.
- from these determinations and body weight data, individual daily food consumption, food efficiency, and mean daily intake of the test substance were calculated.
Gestation and lactation periods:
- individual food consumption of pregnant P1 and F1 females was recorded on gestation days 0, 7, 14, and 21 and on lactation days 0, 7, and 14.
- food consumption was not measured for males or females without evidence of copulation.
- from these determinations and body weight data, individual daily food consumption, food efficiency, and mean daily intake of the test substance were calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (P0 and F1 parental generation): No

CLINCIAL CHEMISTRY
- blood: it was collected from the first 10 rats surviving to scheduled sacrifice in each of the P0 female groups. Collection was done in the morning and plasma was stored.
- plasma samples were analysed for copper, zinc, manganese, and iron concentration. Analysis was done by inductively-coupled plasma (ICP) atomic emission spectroscopy. Due to an instrument malfunction during analysis, no data is available for plasma samples from P0 males.

GESTATION PROCEDURE (from day 20 of gestation for mated females, or from the end of the cohabitation period for females without evidence of copulation):
- female rats were observed at least twice daily for signs of delivery and offspring

Oestrous cyclicity (parental animals):

NOTE: F1 animals selected for mating were named P1 in the result section, since they are considered to be the second parental generation.
- vaginal lavage samples were collected daily from all P0 and F1 female rats in order to determine the stages of the estrous cycle.
- vaginal lavage samples were collected beginning 3 weeks prior to start of cohabitation and continuing until copulation was confirmed or the cohabitation period ended.
- vaginal lavage sample collected on the day copulation was confirmed was not used for estrous cycle evaluation.
- vaginal lavage samples were also collected from all P0 and F1 parental female rats at the time of sacrifice.
- vaginal lavage samples were examined microscopically for determination of the stage of the estrous cycle (diestrus, estrus, proestrus).

Sperm parameters (parental animals):

NOTE: F1 animals selected for mating were named P1 in the result section, since they are considered to be the second parental generation.
Parameters examined in all P0 and F1 male parental generations:
- right epididymis and isolated cauda were weighed. Sperm was obtained from the cauda epididymis and the percentage of motile cells among at least 200 cells examined per animal was determined. Also, an aliquot of the cauda was stained and smears were prepared. Sperm smears were examined to determine the frequency of morphologically abnormal sperm, expressed as percentage of normal cells among at least 200 cells examined per animal.
- the left epididymis and left testis were analysed. The left cauda epididymis was excised, weighed, and homogenized and the left testis was decapsulated and the parenchyma was weighed and homogenized. Sperm count per cauda epididymis and per gram cauda epididymis, and spermatid count per testis and per gram testis were determined.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
- offspring were individually handled and examined for abnormal behavior and appearance on days 0, 4, 7, 14 and 21 postpartum); any dead or abnormal pups were recorded
- day 0 postpartum: live and dead pups in each litter were counted by sex as soon as possible after delivery was completed. Live pups in each litter were individually weighed.
- day 4 postpartum (before standardisation of litters): pups in each litter were counted by sex and individually weighed.
- days 7 and 14 postpartum: pups in each litter were counted by sex and individually weighed.
- day 21 postpartum (weaning): pups in each litter were counted by sex and individually weighed.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Litters of 8 offspring or fewer were not reduced. The number of pups of each sex were recorded.

ASSESSMENT OF DEVELOPMENTAL LANDMARKS (F1 GENERATION):
- vaginal patency: F1 female rats designated for mating were examined for vaginal patency once daily beginning on postnatal day 21 until achievement or postnatal day 43, whichever came first.
- preputial separation: F1 male rats designated for mating were examined for preputial separation beginning on postnatal day 35 until achievement or postnatal day 55, whichever came first.

ASSESSMENT OF DEVELOPMENTAL LANDMARKS (F2 GENERATION):
- anogenital distance: anogenital distance was not recorded in F2 pups on Day 0 postpartum because there were no indications of test substance-related effects on sex ratio or sexual maturation of F1 pups.

CLINICAL CHEMISTRY
- blood: it was collected from the first 10 rats surviving to scheduled sacrifice in each of the F1 and F2 male and female groups. Collection was done in the morning and plasma was stored.
- plasma samples were analysed for copper, zinc, manganese, and iron concentration. Analysis was done by inductively-coupled plasma (ICP) atomic emission spectroscopy. Due to an instrument malfunction during analysis, no data is available for plasma samples from female weanlings.

Postmortem examinations (parental animals):

NOTE: F1 animals selected for mating were named P1 in the result section, since they are considered to be the second parental generation.
SACRIFICE
- Male animals:
P0: test days 109 - 113
F1: test day 119
- Maternal animals:
preganant females (P0 and F1): on day of weaning litters (Day 21 postpartum)
nonpregnant females (P0 and F1): approx. Day 28 after the end of cohabitiation

GROSS NECROPSY
- all P0 and F1 adult rats received a gross pathological examination.
- uteri of all cohabited females were examined for the presence and number of implanation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were collected from all P0 and F1 adult animals and preserved for histopathological examination:
- males: testis, epididymis, prostate, seminal vesicles, and coagulating glands
- females: ovaries, uterus (with oviducts), vagina, and cervix- both sexes: brain, liver, gross observations, kidneys, pancreas, femur, intestine (first 10 cm after stomach), and heart
Reproductive organs, gross observations, and potential target organs (liver and brain) were processed and evaluated microscopically in all control and 1500 mg/kg dose group P0 and F1 adult rats.

- final body weight data, for animals that were sacrificed by design, were used for calculation of organ/body weight ratios.
- P0 and F1 adult rats: weights of the following organs were recorded (paired organs weighed together) for all P0 and F1 adult animals sacrificed by design:
males: testes, epididymis, right cauda epididymis, seminal vesicles (with coagulating glnads, and prostate
females. ovaries and uterus (with oviducts and cervix)
both sexes: liver, brain, kidneys, spleen, adrenal glands, pituitary gland, and thyroid gland
- group means and organ weight ratios were calculated.

OVARIAN FOLLICULAR COUNTS
A quantitative evaluation of primordial and growing follicles was conducted on the first 10 lactating F1 females (surviving to scheduled sacrifice) from control and high-dose (1500 ppm)
groups. Six ovarian cross sections were taken from the central area of the ovary using a step section technique. Primordial and growing follicles (up to but not including antral follicles) were enumerated for up to 12 ovarian sections per animal.

TISSUE METAL CONCENTRATION
- brain and liver samples were analysed for copper, zinc, manganese, and iron concentration. Analysis was done by inductively-coupled plasma (ICP) atomic emission spectroscopy.

Postmortem examinations (offspring):

SACRIFICE/GROSS NECROPSY/ HISTOPATHOLOGY / ORGAN WEIGTHS
- Nursing offspring: pups were euthanized. 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. 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. Organ weights from pups were not recorded.
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age (date of weaning). These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
A gross pathological evaluation was performed on all weanlings with external abnormalities or clinical signs and from one randomly selected pup/sex/litter. All gross lesions and potential target organs (brain and liver) were preserved. Tissues collected at necropsy (liver, brain, and gross observations) were processed and evaluated microscopically from selected (one/sex/litter) F1 and F2 1500 mg/kg dose group and control weanlings.
The following tissues were collected from the same 10 weanlings/sex/dose from which blood was collected (see tissue metal collection below): brain, liver, kidney, pancreas, femur, intestine (first 10 cm of intestine after stomach), and heart. Tissues samples were retained for possible chemical analysis, and additional samples were stored for possible microscopic evaluation.
The liver, brain, spleen, and thymus weights were recorded from one weanling/sex/litter. Group means and organ weight ratios were calculated.

TISSUE METAL CONCENTRATION (F1 and F2 weanlings)
- brain and liver samples were analysed for copper, zinc, manganese, and iron concentration. Analysis was done by inductively-coupled plasma (ICP) atomic emission spectroscopy.

Statistics:

Levene’s test for homogeneity, Shapiro-Wilk test for normality, One-way analysis of variance, Dunnett's test, Kruskal-Wallis test, and Dunn's test: 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, and organ weight
Cochran-Armitage test for trend: incidence of clinical observations, mating index, fertility index, gestation index, and litter survival
Levene’s test for homogeneity, Shapiro-Wilk test for normality, analysis of covariance, Dunnett-Hsu, and non-parametric analysis of covariance: sex ratio (Covariate: litter size), mean pup weights (Covariates: litter size)
For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05.

Reproductive indices:

The following indices of reproductive function were calculated for the P0 and F1 parental animals:
Mating Index (%): (number copulated / number cohabited) X 100
Fertility index (%): (number pregnant / number copulated) X 100
Gestation index (%): (number of litters with at least one live pup / number of litters) X 100
Implantation efficiency (%): (number of pups born / number of implantation sites) X 100

Offspring viability indices:

Pups born alive (%): (number of pups born alive / number of pups born) X 100
Viability index (%): (number of pups alive Day 4 preculling / number of pups born alive) X 100
Lactation index (%): (number of pups alive at weaning (Day 21 postpartum) / number of pups alive Day 4 postculling) X 100
Litter survival (%): number of litters weaned / number of viable litters delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

females: a test substance-related increase in the concentration of copper and a decrease in the concentration of iron were observed in the liver at 1500 ppm (equivalent to 1500 mg/kg).

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS
- males: no test substance-related clinical observations were observed at any dose level
- females: no test substance-related clinical observations were observed during premating, gestation, or lactation at any dose level.

MORTALITY
- males and females: no test substance-related mortality was observed during the study.
- control group: one malewas sacrificed in extremis on day 14 because of a fractured nose.

BODY WEIGHT AND BODY WEIGHT GAIN
- males: no test substance-related effects on body weight or body weight gain were observed at any dose level.
Occasional findings of statistically signifcant increases in body weight gain were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.
- females: no test substance-related effects on body weight or body weight gain during premating, gestation, or lactation at any dose level.
Occasional findings of statistically signifcant increases in body weight gain were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

FOOD CONSUMPTION AND COMPOUND INTAKE
- males: no test substance-related effects on food consumption were observed in P0 males during premating at any dose level.
Occasional findings of statistically significant increases in food consumption were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.
-females: no test substance-related effects on food consumption were observed in P0 females during premating, gestation, or lactation at any dose level.
Occasional findings of statistically significant increases in food consumption were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups (equivalent to 100, 500, 1000 and 1500 mg/kg), respectively, was:
- 1.53, 7.7, 15.2 and 23.6 mg/kg/day for P0 males during premating;
- 1.92, 9.6, 19.1 and 29.5 mg/kg/day for P0 females during premating;
- 1.67, 8.6, 17.0 and 26.2 mg/kg/day for P0 females during gestation;
- 3.39, 17.7, 33.8 and 55.7 mg/kg/day for P0 females during the first 2 weeks of lactation;

FOOD EFFICIENCY
- males: no test substance-related effects on food efficiency were observed in P0 males during premating at any dose level.
The statistically significant decreases in food efficiency in P0 males on days 0-7 and for the entire premating period (days 0-70) at 1500 mg/kg were considered spurious and due to a slightly higher food consumption on days 0-7 in this group.
-females: no test substance-related effects on food efficiency were observed in P0 females during premating, gestation, or lactation at any dose level.
Occasional findings of statistically significant decreases in food efficiency were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

CLINICAL CHEMISTRY
- females: no change in the concentration of copper or iron was observed in the plasma at any dose level. No test substance-related changes in the concentration of manganese or zinc were observed in the plasma.

ORGAN WEIGHTS
Spleen
- small decreases in mean spleen weight parameters were observed in the P1 female adults (9%) (test substance-related and potentially adverse).
- females: a small decrease in the spleen mean organ weight parameters was observed in the 1500 ppm dietary exposure group (equivalent to 1500 mg/kg). Mean absolute and relative (organ wt./body wt.) values were both decreased 9%, as compared to control values. Only the decrease in mean relative spleen weight was statistically significant.
- males: no differences were observed in P1 male spleen weight values.
- all other individual and mean organ weight differences in P1 rats were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
- at the terminal sacrifice, there were no test substance-related gross observations. All gross observations were consistent with normal background lesions in rats of this age and stock.

HISTOPATHOLOGY
- no test substance-related microscopic findings were observed in the liver, brain or reproductive organs. All microscopic findings were considered to be incidental lesions commonly found in rats of this age and stock.
- males: no test substance-related changes in the concentration of copper, iron, manganese or zinc were observed in the liver or brain at any dose level.
- females: no change in the concentration of copper or iron was observed in the brain at any dose level. No test substance-related changes in the concentration of manganese or zinc were observed in the liver or brain.

REPRODUCTION FUNCTION: SPERM MEASURES
- no test substance-related effects were observed on sperm motility, morphology, epididymal sperm or testicular spermatid numbers in P0 males at any dose level.

REPRODUCTION FUNCTION: ESTREOUS CYCLE
- no test substance-related effects were observed on the mean percent days in estrus, diestrus, or proestrus, or mean cycle length in the P0 females at any dose level.
- in P0 females, the mean percent days in estrus at 1000 and 1500 ppm (equivalent to 1000 and 1500 mg/kg) were slightly higher than the control value (47 and 40%, respectively, vs. 30% for the control group). Since the increase was greater at 1000 than 1500 ppm, was not associated with any change in mean estrous cycle length or adverse reproductive outcome, and was not observed in F1 females, it was not considered test substance-related.
- distribution of estrous cycle stages at sacrifice was similar across groups in P0 females.

REPRODUCTIVE PERFORMANCE
- no test substance-related effects were observed on precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency in the P0 generation at any dose level.
- failure of 18 P1 adult pairs to produce litters was not related to test substance exposure. Gross and microscopic evaluation of the 18 pairs revealed a morphological explanation of their infertility in 3 P1 individuals. The cause of the reproductive failure in the remaining 15 pairs was not determined.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The dietary concentration of 1000 mg/kg was equivalent to mean daily intakes of copper of 15.2-23.5 mg/kg bw/day for male rats during premating and 17.0-26.7 mg/kg bw/day for female rats during premating and gestation.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no reproductive effects or toxicity were observed.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg diet

System:

immune system

Organ:

spleen

Treatment related:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- males: concentration of copper was increased in the liver at 1000 and 1500 ppm (equivalent to 1000 and 1500 mg/kg)(possibly test substance-related).
- females: test substance-related increase in the concentration of copper was observed in the liver and brain at 1500 ppm (equivalent to 1500 mg/kg).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
- males: no test substance-related clinical observations were observed at any dose level
- females: no test substance-related clinical observations were observed during premating, gestation, or lactation at any dose level.

MORTALITY
- males and females: no test substance-related mortality was observed during the study.
- 500 ppm (equivalent to 500 mg/kg): one female was sacrificed in extremis on day 109 due to dystocia. another female one was found dead on day 17 due to pyelonephritis. A third female was sacrificed in extremis on day 119 for morbidity of undetermined cause.

BODY WEIGHT AND BODY WEIGHT GAIN
- males: no test substance-related effects on body weight or body weight gain were observed at any dose level.
- females: no test substance-related effects on body weight gain during premating, gestation, or lactation at any dose level.
Occasional findings of statistically signifcant increases in body weight or body weight gain were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

FOOD CONSUMPTION AND COMPOUND INTAKE
- males: no test substance-related effects on food consumption were observed in F1 males during premating.
Occasional findings of statistically significant increases in food consumption were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.
- females: no test substance-related effects on food consumption were observed during premating, gestation or lactation at any dose level.
Occasional findings of statistically significant increases in food consumption were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.


The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups (equivalent to 100, 500, 1000 and 1500 mg/kg), respectively, was:
- 2.25, 11.5, 23.5 and 36.1 mg/kg/day for F1 males during premating;
- 2.65, 13.3, 26.7 and 43.8 mg/kg/day for F1 females during premating;
- 1.69, 8.5, 17.1 and 26.5 mg/kg/day for F1 females during gestation; and
- 3.27, 17.6, 35.2 and 55.4 mg/kg/day for F1 females during the first 2 weeks of lactation

FOOD EFFICIENCY
- males: no test substance-related effects on food efficiency were observed in F1 males during premating.
Occasional findings of statistically significant decreases in food efficiency (due to slightly higher food consumption) were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.
- females: occasional findings of statistically significant decreases in food efficiency (due to slightly higher food consumption) were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

CLINICAL CHEMISTRY:
- males: no test substance-related change in the concentration of copper was observed in the plasma at any dose level. The statistically significant increase in the concentration of copper in the plasma at 1500 ppm was considered spurious due to the small magnitude of the change and considering the variability of the plasma values across groups. No test substance-related changes in the concentration of iron, manganese or zinc were observed in the plasma.
- females: no change in the concentration of copper, iron, manganese or zinc was observed in the plasma at any dose level.

ORGAN WEIGHTS
- male and females: no test substance-related organ weight effects. All individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
- at the terminal sacrifice, there were no test substance-related gross observations. All gross observations were consistent with normal background lesions in rats of this age and stock.

HISTOPATHOLOGY
- no test substance-related microscopic findings were observed in the liver, brain or reproductive organs. All microscopic findings were considered to be incidental lesions commonly found in rats of this age and stock.
- ovarian follicle counts: no significant difference in the total number of primordial and pre-antral follicles between the control and 1500 ppm F1 adult females (equivalent to 1500 mg/kg).
- males: no test substance-related change in the concentration of copper was observed in the brain at any dose level. No test substance-related changes in the concentration of iron, manganese or zinc were observed in the liver or brain. The statistically significant increases in liver zinc concentration at 500 ppm (equivalent to 500 mg/kg) and in copper concentration in the brain at 1000 ppm (equivalent to 1000 mg/kg) were considered spurious since they were not dose-related.
- females: no test substance-related changes in the concentration of iron, manganese or zinc were observed in the liver or brain. The statistically significant increase in manganese concentration in the brain at 1500 ppm (equivalent to 1500 mg/kg) was considered spurious due to the small magnitude of the change and considering the inter-animal variability of across groups. The statistically significant decrease in plasma zinc concentration at 1000 ppm (equivalent to 1000 mg/kg) was considered spurious since it was not dose-related.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P1)

REPRODUCTION FUNCTION: SPERM MEASURES
- no test substance-related effects were observed on sperm motility, morphology, epididymal sperm or testicular spermatid numbers in F1 males at any dose level.

REPRODUCTION FUNCTION: ESTREOUS CYCLE
- no test substance-related effects were observed on the mean percent days in estrus, diestrus, or proestrus, or mean cycle length in the F1 females at any dose level.
- distribution of estrous cycle stages at sacrifice was similar across groups in F1 females.

REPRODUCTIVE PERFORMANCE
- no test substance-related effects were observed on precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency in the F1 generation at any dose level.
- failure of 9 F1 adult pairs to produce litters was not related to test substance exposure. The cause of reproductive failure in one F1 female was dystocia. The cause of the reproductive failure in the remaining 8 pairs was not determined.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: The dietary concentration of 1000 mg/kg was equivalent to mean daily intakes of copper of 15.2-23.5 mg/kg bw/day for male rats during premating and 17.0-26.7 mg/kg bw/day for female rats during premating and gestation.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no reproductive effects or toxicity were observed.

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg diet

System:

immune system

Organ:

spleen

Treatment related:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

- test substance-related increase in the concentration of copper was observed in the liver of F1 male and female weanlings at 1000 and 1500 ppm (equivalent to 1000 and 1500 mg/kg).
- concentration of copper was slightly increased in the brain of F1 male weanlings at 1500 ppm (equivalent to 1500 mg/kg)(possibly test substance-related).

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

CLINICAL SIGNS
- no test substance-related clinical observations were observed in F1 litters at any concentration level.
- clinical observations observed in pups in this study occurred at low incidences and/or were not dose-related.

MORTALITY /VIABILITY
- no test substance-related effects on survival indices during the lactation period at any concentration level in either F1 or F2 litters.

BODY WEIGHT AND WEIGHT GAIN
- no test substance-related effects were observed on F1 pup weights at any dose level.
- increase in pup weight in F1 litters at 100 ppm (equivalent to 100 mg/kg) on lactation day 7 was considered spurious since it was not dose-related.

ORGAN WEIGHTS
- male and female: a small decrease in the spleen mean organ weight parameters was observed in the 1500 ppm dietary exposure group (equivalent to 1500 mg/kg)(not statistically significant). Mean absolute spleen weights were decreased 9% in both sexes, as compared to controls. Mean relative (organ wt./body wt.) spleen weights were decreased 10% and 11% in male and female F1 weanlings, respectively, as compared to controls.
- all other individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
- no test substance-related gross observations in F1 weanlings. Gross observations occurred at low incidences, were randomly distributed across control and treatment groups, and/or were lesions common to rats of this stock and age.
- no test substance-related gross observations in F1 pups. Observations in pups of lungs not expanded and no milk spot in the stomach are nonspecific lesions that are commonly seen in all pups that are born dead (not considered to be test substance related).

HISTOPATHOLOGY
- no test substance-related microscopic findings were observed in the liver and brain of the F1 weanlings. The few lesions present are common spontaneous lesions in this age and stock of rat.
- statistically significant increase in zinc concentration in the liver in F1 male and female weanlings at 1000 and 1500 ppm (equivalent to 1000 and 1500 mg/kg)was considered spurious since it was small and the magnitude of the change was independent of dose.


SEXUAL MATURATION
- no test substance-related effects on the age at preputial separation in F1 males or vaginal opening in F1 females at any dose level.
- in F1 females, the mean age at vaginal opening at 1500 ppm (equivalent to 1500 mg/kg) was significantly increased (33.6 vs. 32.1 days for the control group) but the delay was small (1.5 days) and was within the laboratory’s historical control range. The apparent delay in vaginal opening was not considered test substance-related.

OTHER EFFECTS:
- sex ratio: no test substance-related effects on sex ratio during the lactation period at any concentration level inF1 litters.
- no test substance-related effects on the number of pups born, born alive, or alive on day 4, 7, 14, or 21 at any concentration level in F1 litters.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg diet

System:

immune system

Organ:

spleen

Treatment related:

yes

Relevant for humans:

yes

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- concentration of iron in the plasma was decreased in F2 male and female weanlings at 1500 ppm (equivalent 1500 mg/kg)(possibly test substance-related).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

- test substance-related increase in the concentration of copper was observed in the liver of F2 male and female weanlings at 1000 and 1500 ppm (equivalnet to 1000 and 1500 mg/kg).
- concentration of copper was slightly increased in the brain of F2 male weanlings at 1500 ppm (equivalent to 1500 mg/kg)(possibly test substance-related).

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

CLINICAL SIGNS
- no test substance-related clinical observations were observed in F2 litters at any concentration level.
- clinical observations observed in pups in this study occurred at low incidences and/or were not dose-related.

MORTALITY /VIABILITY
- no test substance-related effects on survival indices during the lactation period at any concentration level in F2 litters.

BODY WEIGHT AND WEIGHT GAIN
- no test substance-related effects were observed on F2 pup weights at any dose level.

CLINICAL CHEMISTRY
- no change in the concentration of copper was observed in the plasma of F2 weanling at any dose level.

ORGAN WEIGHTS
- weanlings had a decrease in mean spleen weight parameters, at the high dose (1500 ppm; equivalent to 1500 mg/kg). Mean absolute spleen weights were decreased 10% and 15% in males and females, respectively, as compared to controls. Mean relative (organ wt./body wt.) spleen weights were also decreased 10% and 15% in males and females, respectively, as compared to controls. Except for the male mean absolute spleen weight decrease (10%), these differences were statistically significant.
- all other individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration.

GROSS PATHOLOGY
- no test substance-related gross observations in F2 weanlings. Gross observations occurred at low incidences, were randomly distributed across control and treatment groups, and/or were lesions common to rats of this stock and age.
- no test substance-related gross observations in F2 pups. Observations in pups of lungs not expanded and no milk spot in the stomach are nonspecific lesions that are commonly seen in all pups that are born dead (not considered to be test substance related).

HISTOPATHOLOGY
- no test substance-related microscopic findings were observed in the liver and brain of the F2 weanlings. The few lesions present are common spontaneous lesions in this age and stock of rat.
- statistically significant increase in manganese concentration in the brain of F2 male and female weanlings at 1500 ppm (equivalent to 1500 mg/kg) was considered spurious due to the small magnitude of the change and considering the inter-animal variability across groups.
- statistically significant increase in zinc concentration in the brain of F2 male weanlings at 1500 ppm (equivalent to 1500 mg/kg) was considered spurious since it was small and was not observed in F2 females or F1 males or females at this dose level.
- statistically significant decrease in zinc concentration in the liver of F2 female weanlings at 100 ppm was considered spurious since it was not dose-related.

OTHER EFFECTS:
- sex ratio: no test substance-related effects on sex ratio during the lactation period at any concentration level in F2 litters.
- no test substance-related effects on the number of pups born, born alive, or alive on day 4, 7, 14, or 21 at any concentration level in F2 litters.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (F2)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg diet

System:

immune system

Organ:

spleen

Treatment related:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (reproductive toxicity): 1500 ppm (equivalent to 1500 mg/kg)
NOAEL (systemic toxicity; parental generations): 1000 ppm (equivalent to 1000 mg/kg; based on reduced spleen weight)
NOAEL (systemic toxicity; offspring generations): 1000 ppm (equivalent to 1000 mg/kg; based on reduced spleen weight)
The dietary concentration of 1000 ppm 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.

In the parental generation, there were no effects considered to be related to copper sulfate pentahydrate treatment on the following parameters at any concentration: clinical signs, mortality, body weight, body weight gain, food consumption, food efficiency, reproductive function/performance, gross pathology, and histopathology (liver, brain and reproductive organs). Furthermore, in the offsprings, there were no effects on sexual maturation, sex ratio, survival, number of pups born, born alive, body weight, clinical observations, gross pathology, and histopathology (liver and brain).
Potentially adverse effects considered to be related to copper sulfate treatment were limited to the 1500 ppm groups and were comprised of decreased sleen weight in P1 adult females , and F1 and F2 male and female weanlings.