Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
See Principles of method
Principles of method if other than guideline:
The following minor deviations occurred from the requirements of OECD guideline No. 416 (adopted 22nd January 2001):
Animal rooms were maintained at a temperature of 18-26ºC instead of the test guideline recommended 19-25%.
The guideline indicates that “Twice daily, during the weekend once daily when appropriate, all animals should be observed for morbundity and mortality. However it is not clear from the report if these intervals were respected. The report indicates that “Cage-site examinations were conducted at least once daily throughout the study”.
Testicular histopathological examinations are not fully described.
These deviations are not considered to have affected the scientific integrity, or outcome of this study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Lot/Batch number: Aldrich Lot 17919TA
Colour: blue.
Physical form: crystal.
Purity: 101.0% (ICP/MS)
Stability: The stability of the test substance over the course of the study was confirmed by purity analyses conducted near the beginning and the end of the study. Analyses were conducted at:
Exygen Research
3058 Research Drive
State College, Pennsylvania 16801
U.S.A.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Laboratories, Inc., Raleigh, North Carolina, US.
Age/weight at study initiation: The P1 generation animals were approximately 8 weeks old at the start of treatment, and in the body weight ranges of approximately 262-332g (males) and approximately 166-231g (females).

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
Start of Cohabitation:
Animals were cohoused after approximately 10 weeks of exposure to the test substance. The day animals were first cohoused was designated as day 1 of cohabitation.

Duration of Cohabitation Period:
Animals were 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.

Evidence of Copulation:
Once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample, either of which was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded. The day evidence of copulation was observed was designated as day 0 of gestation.

Cohousing:
Each female was continually housed on a 1:1 basis with a randomly selected, nonsibling male of the same dietary concentration level, in the male's cage. On the day copulation was confirmed, the female was transferred back to individual cage housing.

Deviations from standard protocol: None.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet preparation analysis demonstrated that the test substance was stable in the diet under study conditions. The homogeneity data support that the mixing procedure was adequate for all dietary levels. The concentration verification data indicate that the test substance was present at the targeted levels during the study.
Duration of treatment / exposure:
Duration of exposure before mating: At least 70 days for both P1 and F1 animals.
See Other information - Table 5.
Details on study schedule:
For treatment and sacrifice schedules, see Other information - Table 5 and Table 6.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000, 1500 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
30 rats/sex/concentration.
See Table 1 ('Table for animal assignment to dosage groups').
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
Clinical signs:
Cage-site examinations were conducted at least once daily throughout the study. Moribund rats were sacrificed. At least once weekly throughout the premating feeding, gestation, and lactation periods, each of the P1 and F1 parental rats was individually handled and carefully examined for abnormal behavior and/or appearance.

Body weight:
Premating Period: All P1 and F1 rats were weighed once a week. All rats evaluated for developmental landmarks (vaginal patency, preputial separation) were weighed on the day of achievement.
Gestation and Lactation Periods: P1 and F1 dams were weighed on days 0, 7, 14, and 21 of each period. Females without evidence of copulation, those that copulated and did not deliver a litter, and males were weighed on a weekly schedule.

Food consumption:
Premating and Cohabitation Periods: Individual food consumption was determined weekly for all P1 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.
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:
Tap water was provided ad libitum. Water consumption was not measured.
Oestrous cyclicity (parental animals):
Vaginal lavage samples were collected daily from all P1 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. The 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 P1 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):
Sperm parameters for all P1 and F1 parental males were evaluated. At sacrifice, the right epididymis was weighed, the cauda was removed, weighed, and placed in 32°C phosphate buffered saline with 10 mg/mL bovine serum albumin, pricked with a needle to facilitate the release of sperm, and placed in an incubator at 35°C for 5 minutes. After incubation, an aliquot was placed in a sample chamber which was loaded into a Hamilton Thorne Integrated Visual Optical System (IVOS). The percentage of motile cells among at least 200 cells examined per animal was determined.

The right cauda epididymis was further incubated at 35°C for approximately 15 minutes. An aliquot was stained with eosin, and smears were prepared on microscope slides. 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 specific types of abnormal morphologies, if any, were not categorized. The excised right cauda and the right caput and corpus epididymis were then placed in Bouin’s solution.

The left epididymis and left testis were flash frozen in liquid nitrogen and stored at –65° to –85°C until analyzed. After thawing, the cauda epididymis was excised, weighed, and homogenized. After thawing, the 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 using the IVOS.
Litter observations:
Lactation Procedures:
The day when delivery was complete was designated day 0 postpartum. At each examination period (days 0, 4, 7, 14, and 21 postpartum), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. Dams that had no live pups remaining during lactation were sacrificed.

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:
Pups in each litter were counted by sex and individually weighed. Then, litters were culled randomly to 8 (4/sex when possible) and the number of pups of each sex recorded. Extra offspring were euthanized (by decapitation) and discarded without pathological examination. Litters of 8 offspring or fewer were not reduced.

Days 7 and 14 Postpartum:
Pups in each litter were counted by sex and individually weighed.

Day 21 Postpartum (Weaning - Postnatal day 21):
Pups in each litter were counted by sex and individually weighed. Offspring in the F1 litters of each treatment level were randomly selected (one rat/sex/litter when possible) to serve as parents for the F2 generation and were placed in individual cages. For groups without sufficient litters, additional pups were chosen from randomly selected litters within the group to achieve the required group size. Selection of rats within litters was random.
Postmortem examinations (parental animals):
Organ Weights:

Weights of the following organs were recorded (paired organs weighed together) for all P1 adult animals sacrificed by design. Group means and organ weight ratios (organ wt./final body wt. and organ wt./brain wt.) were calculated.

Male: Testes, Epididymides, Right Cauda Epididymis, Seminal Vesicles (with coagulating glands), Prostate.
Female: Ovaries, Uterus (with oviducts and cervix).
Both Sexes: Liver, Brain, Kidneys, Spleen, Adrenal Glands, Pituitary Gland, Thyroid Gland.
(Pituitary and thyroid glands were weighed after fixation.)


Histopathology:

Tissues designated for microscopic evaluation were embedded in paraffin, cut at a nominal thickness of 5 micrometers, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.

P1 Adult Rats:
Reproductive organs, gross observations, and potential target organs (liver and brain) were processed and evaluated microscopically in all control and high-dose P1 adult rats. Tissue from rats in the low- and intermediate P1 adult dose groups did not require examination to determine a no-observed-adverse-effect level.

In addition, the reproductive organs from all mated animals that failed to produce a litter (i.e. reproductive failures) were evaluated microscopically. These included 18 P1 pairs.

Most gross lesions in P1 adults were saved and evaluated microscopically. Selected gross observations for which a microscopic diagnosis would not be additive (e.g. osteoarthritis, pododermatitis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinnae) were saved, but not processed for microscopic evaluation.
Postmortem examinations (offspring):
Organ Weights:

Nursing Offspring:
Organ weights from pups were not recorded.

F1 and F2 Weanlings:
The liver, brain, spleen, and thymus weights were recorded from one weanling/sex/litter. Group means and organ weight ratios (organ wt./final body wt. and organ wt./brain wt.) were calculated.

Final body weight data, for animals that were sacrificed by design, were used for calculation of organ/body weight ratios.

Adult F1 Offspring:
Weights of the following organs were recorded (paired organs weighed together) for all F1 adult animals sacrificed by design. Group means and organ weight ratios (organ wt./final body wt. and organ wt./brain wt.) were calculated.

Male: Testes, Epididymides, Right Cauda Epididymis, Seminal Vesicles (with coagulating glands), Prostate.
Female: Ovaries, Uterus (with oviducts and cervix).
Both Sexes: Liver, Brain, Kidneys, Spleen, Adrenal Glands, Pituitary Gland, Thyroid Gland.
(Pituitary and thyroid glands were weighed after fixation.)

Histopathology:

Tissues designated for microscopic evaluation were embedded in paraffin, cut at a nominal thickness of 5 micrometers, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.

Nursing Offspring:
Microscopic examination of tissues from pups (nursing offspring) that died (found dead, sacrificed in extremis, or accidentally killed) during the lactation period was not performed.

F1 and F2 Weanlings:
Tissues collected at necropsy (liver, brain, and gross observations) were processed and evaluated microscopically from selected (one/sex/litter) F1 and F2 high-dose and control weanlings. Examination of tissues from other groups was not required to establish a no-observed-adverse-effect level.

F1 Adult Rats
Reproductive organs, gross observations, and potential target organs (liver and brain) were processed and evaluated microscopically in all control and high-dose F1 adult rats. Tissue from rats in the low- and intermediate F1 adult dose groups did not require examination to determine a no-observed-adverse-effect level.

In addition, the reproductive organs from all mated animals that failed to produce a litter (i.e. reproductive failures) were evaluated microscopically. These included 9 F1 pairs.

Most gross lesions in F1 adults were saved and evaluated microscopically. Selected gross observations for which a microscopic diagnosis would not be additive (e.g. osteoarthritis, pododermatitis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinnae) were saved, but not processed for microscopic evaluation.
Reproductive indices:
For indices calculated, see Other information below.
Offspring viability indices:
For indices calculated, see Other information below.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

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)

Parent Males (P1 Males):

Intake of Test Substance:
The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups, respectively, was:
1.53, 7.7, 15.2 and 23.6 mg/kg/day for P1 males during premating.

Clinical Observations and Mortality:
There was no test substance-related mortality during the study. No test substance-related clinical observations were observed at any dose level.

Mean Body Weights and Body Weight Gains:
No test substance-related effects on body weight or body weight gain were observed at any dose level. Occasional findings of statistically significant 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 Food Efficiency:
There were no test substance-related effects on food consumption or food efficiency in P1 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. The statistically significant decreases in food efficiency in P1 males on days 0-7 and for the entire premating period (days 0-70) at 1500 ppm were considered spurious and due to a slightly higher food consumption on days 0-7 in this group.

Sperm Parameters:
There were no test substance-related effects on sperm motility, morphology, epididymal sperm or testicular spermatid numbers at any dose level.

Reproductive Indices and Precoital Interval:
The following parameters (where relevant) were not affected by test substance administration: precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency at any dose level.

Cause of Death:
There were no test substance-related deaths in the study. Of the 120 P1 adult males, one animal was sacrificed in extremis on day 14 because of a fractured nose.

Organ Weight Data:
There were no treatment related organ weight changes.

Gross Findings:
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.

Microscopic Findings:
In P1 adult rats, there were no test substance-related microscopic findings 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.

P1 Adult Reproductive Failures:
The failure of 18 P1 adult pairs to produce litters was not related to test substance exposure. Gross and microscopic evaluation revealed a morphological explanation of their infertility in 3 P1 individuals (absence of recent corpora lutea). The cause of the reproductive failure in the remaining pairs was not determined.


Parent Females:

Intake of Test Substance:
The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups, respectively, was:

1.92, 9.6, 19.1 and 29.5 mg/kg/day for P1 females during premating;

1.67, 8.6, 17.0 and 26.2 mg/kg/day for P1 females during gestation;

3.39, 17.7, 33.8 and 55.7 mg/kg/day for P1 females during the first 2 weeks of lactation.

Clinical Observations and Mortality:
There was no test substance-related mortality during the study. No test substance-related clinical observations were observed during premating, gestation, or lactation at any dose level.

Mean Body Weights and Body Weight Gains:
There were 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 significant decreases in body weight gain were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

Food Consumption and Food Efficiency:
There were no test substance-related effects on food consumption or food efficiency in P1 females during premating, gestation, or lactation at any dose level. Occasional findings of statistically significant increases in food consumption or decreases in food efficiency were considered spurious due to their small magnitude, sporadic nature and/or lack of dose-response relationship.

Estrous Cycle Parameters:
There were no test substance-related effects on the mean percent days in estrus, diestrus, or proestrus, or mean cycle length at any dose level.

In P1 females, the mean percent days in estrus at 1000 and 1500 ppm 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.

The distribution of estrous cycle stages at sacrifice was similar across groups.

Reproductive Indices and Precoital Interval:
The following parameters (where relevant) were not affected by test substance administration: precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency at any dose level.

Cause of Death:
There were no deaths amongst P1 adult females.

Organ Weight Data P1 adult rats (see Table 2):
In P1 female adult rats, there was a small decrease in the spleen mean organ weight parameters in the 1500 ppm dietary exposure group. 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. 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 Findings:
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.

Microscopic Findings:
In P1 adult rats, there were no test substance-related microscopic findings 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.

P1 Adult Reproductive Failures:
The failure of 18 P1 adult pairs to produce litters was not related to test substance exposure. Gross and microscopic evaluation revealed a morphological explanation of their infertility in 3 P1 individuals. (absence of recent corpora lutea). The cause of the reproductive failure in the remaining pairs was not determined.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
> 1 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproductive toxicity was seen at any concentration.
Dose descriptor:
LOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased spleen weight in P1 adult females. No reproductive toxicity was seen at any concentration.
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Equivalent to 23.6 mg Cu/kg bw/day for P1 males during premating.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Equivalent to 19.1, 17.0 and 33.8 mg Cu/kg bw/day for P1 females during premating, gestation and the first 2 weeks of lactation, respectively.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed

Details on results (F1)

F1 Males:

Intake of Test Substance (also see Other information)
The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups, respectively, was:
2.25, 11.5, 23.5 and 36.1 mg/kg/day for F1 males during premating.

Clinical Observations and Mortality:
There was no test substance-related mortality during the study. No test substance-related clinical observations were observed at any dose level.

Mean Body Weights and Body Weight Gains:
No test substance-related effects on body weight or body weight gain were observed at any dose level.

Food Consumption and Food Efficiency:
There were no test substance-related effects on food consumption or food efficiency in F1 males during premating. Occasional findings of statistically significant increases in food consumption and 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.

Sperm Parameters:
There were no test substance-related effects on sperm motility, morphology, epididymal sperm or testicular spermatid numbers at any dose level.

Reproductive Indices and Precoital Interval:
The following parameters (where relevant) were not affected by test substance administration: precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency at any dose level.

F1 Offspring Data:
Litter Size, Sex Ratio and Pup Survival: There were no test substance-related effects on the number of pups born, born alive, alive on day 4, 7, 14, or 21, nor were there any effects on sex ratio, or survival indices during the lactation period at any concentration level.

Clinical Observations in pups: There were no test substance-related clinical observations 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.

Pup Weights: No test substance-related effects were observed on F1 pup weights at any dose level. The increase in pup weight in F1 litters at 100 ppm on lactation day 7 was considered spurious since it was not dose-related.

Developmental Landmarks:
There were no test substance-related effects on the age at preputial separation in F1 males at any dose level.

Cause of Death:
There were no deaths amongst F1 adult males.

Organ Weight Data:
F1 Adult Rats: In F1 male (and female) adult rats, there were 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.
F1 Weanlings (see Table 3): In F1 male (and female) weanlings, there was a small decrease in the spleen mean organ weight parameters in the 1500 ppm dietary exposure group, that was 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 Findings:
F1 Adult Rats: 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.
F1 Weanlings: There were 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.
F1 Pups: There were 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, and thus are not considered to be test substance related.

Microscopic Findings:
F1 Adult Rats: In F1 adult rats, there were no test substance-related microscopic findings 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.
F1 Weanlings: There were no test substance-related microscopic findings in the liver and brain of the F1 weanlings. The few lesions present (amongst F1 and F2 weanlings) are common spontaneous lesions in this age and stock of rat.

F1 Adult Reproductive Failures:
The 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 pairs was not determined.


F1 Females:

Intake of Test Substance (also see Other information):
The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups, respectively, was:

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;

3.27, 17.6, 35.2 and 55.4 mg/kg/day for F1 females during the first 2 weeks of lactation.

Clinical Observations and Mortality:
There was no test substance-related mortality during the study. No test substance-related clinical observations were observed during premating, gestation, or lactation at any dose level.

Mean Body Weights and Body Weight Gains:
There were no test substance-related effects on body weight gain during premating, gestation, or lactation at any dose level. Occasional findings of statistically significant 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 Food Efficiency:
There were no test substance-related effects on food consumption during premating, gestation or lactation at any dose level. Occasional findings of statistically significant increases in food consumption and 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.

Estrous Cycle Parameters:
There were no test substance-related effects on the mean percent days in estrus, diestrus, or proestrus, or mean cycle length any dose level. The distribution of estrous cycle stages at sacrifice was similar across groups.

Reproductive Indices and Precoital Interval:
The following parameters (where relevant) were not affected by test substance administration: precoital interval length, mating, fertility, gestation length, number of implantation sites, or implantation efficiency at any dose level.

F1 Offspring Data:
Litter Size, Sex Ratio and Pup Survival: There were no test substance-related effects on the number of pups born, born alive, alive on day 4, 7, 14, or 21, nor were there any effects on sex ratio, or survival indices during the lactation period at any concentration level.

Developmental Landmarks
There were no test substance-related effects on the age at vaginal opening in F1 females at any dose level. In F1 females, the mean age at vaginal opening at 1500 ppm 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 (see below). Therefore, the apparent delay in vaginal opening was not considered test substance-related.

Historical Control Data for Haskell Laboratory 1999-2003:
Study Start Date Mean Day of Vaginal Opening
24-Sep-03 32.3
26-Sep-02 31.3
24-Apr-02 33.9
15-Oct-02 31.3
15-Mar-02 31.7
20-Feb-02 33.0
15-Mar-01 31.4
12-Dec-00 32.1
18-Sep-00 32.3
29-Jul-99 32.8
17-Jun-99 32.1
8-Apr-99 33.1
15-Mar-99 32.5

Mean 32.3
Standard Deviation 0.8
Minimum 31.3
Maximum 33.9

Ovarian Follicle Counts:
There was no significant difference in the total number of primordial and pre-antral follicles between the control and 1500 ppm F1 adult females.

Cause of Death:
There were no test substance-related deaths in the study.
Of the 120 F1 females the following premature deaths occurred:
One animal from the 500 ppm group was sacrificed in extremis on day 109 due to dystocia;
One animal from the 500 ppm group was found dead on day 17 due to pyelonephritis;
One animal from the 1000 ppm group was sacrificed in extremis on day 119 for morbidity of undetermined cause.

Organ Weight Data:
F1 Adult Rats: In F1 female (and male) adult rats, there were 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.
F1 Weanlings (see Table 3): In F1 female (and male) weanlings, there was a small decrease in the spleen mean organ weight parameters in the 1500 ppm dietary exposure group, that was 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 Findings:
F1 Adult Rats: 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.
F1 Weanlings: There were 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.
F1 Pups: There were 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, and thus are not considered to be test substance related.

Microscopic Findings:
F1 Adult Rats: In F1 adult rats, there were no test substance-related microscopic findings 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.
F1 Weanlings: There were no test substance-related microscopic findings in the liver and brain of the F1 weanlings. The few lesions present (amongst F1 and F2 weanlings) are common spontaneous lesions in this age and stock of rat.

F1 Adult Reproductive Failures:
The 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 pairs was not determined.


F2 Males:

F2 Offspring Data:
Litter Size, Sex Ratio and Pup Survival:
There were no test substance-related effects on the number of pups born, born alive, alive on day 4, 7, 14, or 21, nor were there any effects on sex ratio, or survival indices during the lactation period at any concentration level.

Clinical Observations in pups:
There were no test substance-related clinical observations 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.

Pup Weights:
No test substance-related effects were observed on F2 pup weights at any dose level.

Organ Weight Data:
F2 Weanlings (see Table 4): The F2 weanlings had a decrease in mean spleen weight parameters, at the high dose (1500 ppm), that was similar to that observed in the F1 weanlings. 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 Findings:
F2 Weanlings: There were 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.
F2 Pups: There were 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, and thus are not considered to be test substance related.

Microscopic Findings:
F2 Weanlings: There were no test substance-related microscopic findings in the liver and brain of the 21 weanlings. The few lesions present (amongst F1 and F2 weanlings) are common spontaneous lesions in this age and stock of rat.


F2 Females:

F2 Offspring Data:
Litter Size, Sex Ratio and Pup Survival:
There were no test substance-related effects on the number of pups born, born alive, alive on day 4, 7, 14, or 21, nor were there any effects on sex ratio, or survival indices during the lactation period at any concentration level.

Clinical Observations in pups:
There were no test substance-related clinical observations 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.

Pup Weights:
No test substance-related effects were observed on F2 pup weights at any dose level.

Organ Weight Data:
F2 Weanlings (see Table 4): The F2 weanlings had a decrease in mean spleen weight parameters, at the high dose (1500 ppm), that was similar to that observed in the F1 weanlings. 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 Findings:
F2 Weanlings: There were 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.
F2 Pups: There were 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, and thus are not considered to be test substance related.

Microscopic Findings:
F2 Weanlings: There were no test substance-related microscopic findings in the liver and brain of the 21 weanlings. The few lesions present (amongst F1 and F2 weanlings) are common spontaneous lesions in this age and stock of rat.

Effect levels (F1)

open allclose all
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased spleen weight in F1 male weanlings. No reproductive toxicity was seen at any concentration.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased spleen weight in F1 female weanlings. No reproductive toxicity was seen at any concentration.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Effects were seen in F1 weanlings. Equivalent to 23.5 mg Cu/kg bw/day for adults at 1000 ppm.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Effects were seen in F1 weanlings. 1000 ppm is equivalent to 26.7, 17.1 and 35.2 mg Cu/kg bw/day for F1 females during premating, gestation and the first 2 weeks of lactation, respectively.

Results: F2 generation

Effect levels (F2)

open allclose all
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased spleen weight in F2 male weanlings.
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased spleen weight in F2 female weanlings.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Effects were seen in F2 weanlings.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Effects were seen in F2 weanlings.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Intake of Test Substance

The overall mean daily intake of copper in the 100, 500, 1000 and 1500 ppm groups, respectively, was:

1.53, 7.7, 15.2 and 23.6 mg/kg/day for P1 males during premating;

1.92, 9.6, 19.1 and 29.5 mg/kg/day for P1 females during premating;

1.67, 8.6, 17.0 and 26.2 mg/kg/day for P1 females during gestation;

3.39, 17.7, 33.8 and 55.7 mg/kg/day for P1 females during the first 2 weeks of lactation;

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.

Tissue Metal Concentrations

P1 Male Rats

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.  Plasma concentration data was not available for P1 males; however, this had no impact on the study since plasma data was available for P1 females, F1 males and females, and F2 male and female weanlings.  The statistically significant decrease in liver iron concentration at 1500 ppm was considered spurious since there was high interindividual animal variability across groups for this parameter and because the change did not correlate, as in P1 females, with increased liver copper concentration.

P1 Female Rats

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.  No change in the concentration of copper or iron was observed in the plasma or brain at any dose level.

No test substance-related changes in the concentration of manganese or zinc were observed in the liver, plasma or brain.

 

F1 Male Rats

The concentration of copper was increased in the liver at 1000 and 1500 ppm.  Although of small magnitude compared to the increase observed in P1 and F1 females, the increase was considered possibly test substance-related since a few animals in these groups had concentrations that were 2-3 fold higher than the control group mean. However, this is an expected physiological response of homeostatic control and the absence of liver pathology (histological data) confirms that this is not an adverse effect.  No test substance-related change in the concentration of copper was observed in the plasma or brain 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 liver, plasma or brain.  The statistically significant increases in liver zinc concentration at 500 ppm and in copper concentration in the brain at 1000 ppm were considered spurious since they were not dose-related.

 

F1 Female Rats

A test substance-related increase in the concentration of copper was observed in the liver and brain at 1500 ppm.  No change in the concentration of copper was observed in the plasma at any dose level.

No test substance-related changes in the concentration of iron, manganese or zinc were observed in the liver, plasma or brain.  The statistically significant increase in manganese concentration in the brain at 1500 ppm 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 was considered spurious since it was not dose-related.

 

F1 and F2 Weanlings

A test substance-related increase in the concentration of copper was observed in the liver of F1 and F2 male and female weanlings at 1000 and 1500 ppm.  The concentration of copper was slightly increased in the brain of F1 and F2 male (but not female) weanlings at 1500 ppm, and was considered possibly test substance-related.  No change in the concentration of copper was observed in the plasma of F2 weanling at any dose level.  Plasma concentration data was not available for the F1 weanlings; however, this had no impact on the study since plasma data was available for P1 females, F1 males and females, and F2 male and female weanlings.

The concentration of iron in the plasma was decreased in F2 male and female weanlings at 1500 ppm and was considered possibly test substance-related.

The statistically significant increase in manganese concentration in the brain of F2 male and female weanlings at 1500 ppm was considered spurious due to the small magnitude of the change and considering the inter-animal variability across groups.  The statistically significant increase in zinc concentration in the liver in F1 male and female weanlings at 1000 and 1500 ppm was considered spurious since it was small and the magnitude of the change was independent of dose. The statistically significant increase in zinc concentration in the brain of F2 male weanlings at 1500 ppm was considered spurious since it was small and was not observed in F2 females or F1 males or females at this dose level.  The 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.

Table 2. Mean Absolute Spleen Weights (grams) in Male and Female P1 Adult Rats.

 

 

Males

Females

Concentration (ppm)

0

100

500

1000

1500

0

100

500

1000

1500

Final Body

595.4

600.1

603.9

599.5

586.8

328.8

332.2

335.8

333.3

331.9

Spleen

0.866

0.887

0.892

0.881

0.841

0.643

0.629

0.639

0.605

0.586

- underlined values were interpreted to be test substance-related organ weight effects.

 

 

Table 3. Mean Absolute Spleen Weights in Male and Female F1 Weanlings.

 

 

Males

Females

Dose (ppm)

0

100

500

1000

1500

0

100

500

1000

1500

Weight (grams)

Final Body

58.3

60.1

60.9

56.6

58.7

54.5

56.8

56.2

53.5

55.3

Spleen

0.256

0.290

0.280

0.238

0.232

0.245

0.283*

0.265

0.236

0.223

- underlined values were interpreted to be test substance-related organ weight effects.

* statistically significant increase (parametric comparison to control: Dunnett/Tamhane-Dunnett Test)

 

 

Table 4. Mean Absolute Spleen Weights in Male and Female F2 Weanlings.

 

 

Males

Females

Dose (ppm)

0

100

500

1000

1500

0

100

500

1000

1500

Weight (grams)

Final Body

56.9

59.3

59.2

59.8

57.3

54.6

56.8

56.8

55.3

54.7

Spleen

0.253

0.269

0.254

0.252

0.227

0.254

0.265

0.252

0.243

0.217*

- underlined values were interpreted to be test substance-related organ weight effects.

* statistically significant decrease (parametric comparison to control: Dunnett/Tamhane-Dunnett Test)

 

Applicant's summary and conclusion

Conclusions:
LO(A)EL:
Parent Males: No effects up to 1500 ppm. No reproductive toxicity was seen at any concentration.
Parent Females: 1500 ppm (decreased spleen weight in P1 adult females). No reproductive toxicity was seen at any concentration.
F1 Males: 1500 ppm (decreased spleen weight in F1 male weanlings). No reproductive toxicity was seen at any concentration.
F1 Females: 1500 ppm (decreased spleen weight in F1 female weanlings). No reproductive toxicity was seen at any concentration.
F2 Males: 1500 ppm (decreased spleen weight in F2 male weanlings).
F2 Females: 1500 ppm (decreased spleen weight in F2 female weanlings).

NO(A)EL:
Parent Males: 1500 ppm. Equivalent to 23.6 mg/kg bw/day for P1 males during premating.
Parent Females: 1000 ppm. No reproductive toxicity was seen at any concentration. Equivalent to 19.1, 17.0 and 33.8 mg/kg bw/day for P1 females during premating, gestation and the first 2 weeks of lactation, respectively.
F1 Males: 1000 ppm. No reproductive toxicity was seen at any concentration. Effects were seen in F1 weanlings. (See Other information on results for the mg/kg bw/day equivalent for adults at 1000 ppm.)
F1 Females: 1000 ppm. No reproductive toxicity was seen at any concentration. Effects were seen in F1 weanlings. (See Other information on results for the mg/kg bw/day equivalent for adults at 1000 ppm.)
F2 Males: 1000 ppm. No reproductive toxicity was seen at any concentration. Effects were seen in F2 weanlings. (See Other information on results for the mg/kg bw/day equivalent for adults at 1000 ppm.)
F2 Females: 1000 ppm. No reproductive toxicity was seen at any concentration. Effects were seen in F2 weanlings. (See Other information on results for the mg/kg bw/day equivalent for adults at 1000 ppm.)
Executive summary:

Materials and Methods

A two-generation reproduction study, which involved the production of one set of litters in each generation, was conducted with copper sulphate pentahydrate.  Throughout the study, Crl:CD®(SD)IGS BR rats (30/sex/concentration) were fed diets containing 0, 100, 500, 1000 or 1500 ppm copper sulfate.  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. Brain and liver samples were collected from culled pups on postnatal day 4 (12 per group, randomly selected) for possible analysis of copper, zinc, manganese and iron concentrations; analysis was not performed since it was not considered necessary to meet the objectives of the study.

Clinical observations, body weight, and food consumption were determined weekly throughout the study.  Litter examinations (number of live and dead, individual pup weights, clinical observations) were determined at birth, on day 4, and weekly during the lactation period.  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.  The age at either vaginal opening or preputial separation was recorded for the F1 generation.  Sperm motility, morphology, and concentration in the cauda epididymis, and spermatid concentration in the testis were determined for P1 and F1 rats.

At weaning, a gross postmortem examination was performed on one weanling/sex/litter from F1 and F2 litters and gross lesions were retained; the liver, brain, spleen, and thymus were weighed and gross lesions and target organs (brain, liver) retained.  After litter production, all P1 and F1 rats were given a gross pathological examination and the testes, epididymides, right cauda epididymis, seminal vesicles (with coagulating glands and their fluids), prostate, ovaries, and uterus (with oviducts and cervix), thyroid gland, brain, liver, spleen, adrenal glands, pituitary, and kidneys were weighed.  The right testis and epididymis, prostate, seminal vesicles (with coagulating glands), pituitary, ovaries, uterus (with oviducts and cervix), vagina, brain, liver and gross lesions were placed in fixative for all P1 and F1 adults.  These tissues as well as target organs and gross lesions from F1 weanlings in the control and 1500 ppm groups were examined microscopically; tissues in the low and intermediate groups were subsequently evaluated as needed to determine a no-adverse-effect level.  Reproductive organs from the low and intermediate group P1 and F1 rats with suspected reduced fertility were examined microscopically.  Ovarian follicle numbers were evaluated in control and 1500 ppm F1 rats (10/sex/group).

Blood and selected tissues (brain, liver, kidney, pancreas, femur, intestine and heart) were collected from P1 and F1 adults and F1 and F2 weanlings (10/sex/group); plasma and tissues were stored frozen for possible analysis of copper, zinc, manganese and iron concentrations. In addition, selected tissues (kidney, pancreas, femur, intestine and heart) from the same animals were placed in fixative for possible microscopic examination; evaluation was not performed since it was not considered necessary to meet the objectives of the study. The concentration of copper, zinc, manganese and iron was determined in all plasma, brain and liver samples; analysis of other tissues (kidney, pancreas, femur, intestine and heart) was not performed since it was not considered necessary to meet the objectives of the study.

This study was conducted in compliance with OECD test guideline 416 (adopted 22ndJanuary 2001) with the exception of minor deviations (see Principles of method).  These deviations are not considered to have affected the scientific integrity, or outcome of this study.

 

 

Results and Discussion

The mean achieved dose levels of copper for P1 and F1 generation parental animals of both sexes were within the ranges 1.53-2.65, 7.7-13.3, 15.2-26.7 and 23.6-43.8 mg/kg body weight/day, for the 100, 500, 1000 and 1500 ppm groups, respectively. During the first 2 weeks of lactation, achieved dose levels exceeded these ranges in lactating dams.

The concentration of copper was increased in the liver of F1 males and F1 and F2 male and female weanlings at 1000 and 1500 ppm and in P1 and F1 females at 1500 ppm. Brain copper concentration was increased in F1 females and F1 and F2 male weanlings at 1500 ppm. The concentration of liver iron was decreased in P1 females at 1500 ppm. The concentration of plasma iron was decreased in F2 male and female weanlings at 1500 ppm.

There were no effects considered to be related to copper sulfate treatment on the following parameters at any concentration:

  • Mortality and clinical signs of toxicity in P1 and F1 males and females.
  • Body weights, weight gain, food consumption, food efficiency in P1 and F1 males and females.
  • Sperm and estrous cycle parameters in P1 and F1 males and females.
  • Mating, precoital interval, fertility, gestation length, number of implantation sites, and implantation efficiency in the P1 and F1 generations.
  • Number of pups born, born alive, alive on day 4, 7, 14, or 21, sex ratio, and survival indices during the lactation period in F1 and F2 litters.
  • Body weights and clinical observations in F1 and F2 litters during lactation
  • Age at preputial separation in F1 males and vaginal opening in F1 females.
  • Ovarian follicle counts in F1 females.
  • Weight of testes, epididymides, right cauda epididymis, seminal vesicles, prostate, ovaries, uterus, thyroid gland, brain, liver, adrenal glands, kidneys and pituitary in P1 and F1 males and females.
  • Weight of the spleen in P1 males and F1 males and females.
  • Weight of liver, brain and thymus in F1 and F2 weanlings.
  • Gross observations in P1 and F1 adults and F1 and F2 weanlings.
  • Microscopic observations in the liver, brain and reproductive organs in P1 and F1 adults.
  • Microscopic observations in the liver and brain in F1 and F2 weanlings.

Potentially adverse effects considered to be related to copper sulfate treatment were limited to the 1500 ppm groups and were comprised of:

  • Decreased spleen weight in P1 adult females, and F1 and F2 male and female weanlings (see discussion below).

 

Discussion on spleen weight changes:

The small (9%-15%) decrease in mean spleen weight parameters in weanlings may have been spurious but was considered most likely test substance related since it was observed in both the F1 and F2 male and female weanlings.  This is a conservative interpretation of the data, considering the highly variable nature of weanling spleen weights as illustrated by the clearly spurious, statistically significant, increase (16%) in the mean absolute splenic weight of the low-dose F1 female weanlings.

Since weanling spleens were not examined microscopically, the decrease in spleen weights were considered potentially adverse.  Nonetheless, the following would suggest that the decrease in weanling spleen weights may have been a transient physiological alteration such as a marginal decrease in sinusoidal dilatation:

  • The ranges of high-dose weanling splenic weights were similar to the respective control ranges. Of the 111 high-dose weanlings, only 4 had spleen weights below the range of their respective control group, and these were within the general range of weanling spleen weights.
  • There was no test substance-related effect on thymus weight, suggesting that the lymphoid system was not affected by the test substance.
  • All control and high-dose weanling livers had the normal amount of extramedullary hematopoiesis in the livers (microscopic examination), suggesting that the hematopoietic system was not affected by the test substance.
  • There were no test substance-related effects observed in F1 adults.

 

Similarly, the small (9%) decrease in mean spleen weight parameters in the P1 female adults was also interpreted to be test substance related and potentially adverse.  Since spleens were not collected at necropsy, microscopic evaluation of these spleens was also not conducted.

Under the conditions of this study, the no-observed-effect level (NOEL)a for reproductive toxicity was 1500 ppm, the highest concentration tested.  The NOELa for P1 and F1 rats and F1 and F2 offspring during lactation was 1000 ppm, based on reduced spleen weight in P1 adult females, and F1 and F2 male and female weanlings at 1500 ppm however the transient reduced spleen weights are not considered a reproductive endpoint as it did not affect growth or fertility.

In compliance with the “Definition of reproductive toxicity”, OECD document ENV/JM/MONO(2001)6 the spleen effect cannot be considered a reproductive effect as this must include:

  • Adverse effects on sexual function and fertility in adult males and females.
  • Developmental toxicity in the offspring.

For a compound to be considered to be a reproductive toxin “data for animal studies ideally should provide clear evidence of specific reproductive toxicity in the absence of other, systemic, toxic effects”.

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.

aThe study report indicates that the NOEL for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected.  Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency and to the no-observed-adverse-effect level (NOAEL) as defined by the European Union.