Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
other: 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
Details on test animals and environmental conditions:
Source: Charles River Laboratories, Inc., Raleigh, North Carolina, US.
Age/weight at study initiation: The P1 females were approximately 8 weeks old at the start of treatment and in the body weight range of approximately 166-231g.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
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.
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.
Duration of treatment / exposure:
Duration of exposure before mating: At least 70 days for both P1 and F1 animals.
See Other information - Table 5.
Duration of test:
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

Maternal 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 and those that copulated and did not deliver a litter, 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.
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 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.

Organ Weights:
Weights of the following organs were recorded (paired organs weighed together) for all P1 adult animals sacrificed by design: Ovaries, Uterus (with oviducts and cervix), Liver, Brain, Kidneys, Spleen, Adrenal Glands, Pituitary Gland, Thyroid Gland (Pituitary and thyroid glands were weighed after fixation.). Group means and organ weight ratios (organ wt./final body wt. and organ wt./brain wt.) were calculated.

Oestrous cyclicity:
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).

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 and 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 P1 and F1 adult rats. Tissue from rats in the low- and intermediate P1 and 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 18 P1 pairs and 9 F1 pairs.

Most gross lesions in P1 and 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.
Ovaries and uterine content:
See "other information", below
Fetal examinations:
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.

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.

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.
Indices:
See "other information", below.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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. All other individual and mean organ weight differences in P1 rats were considered to be spurious and unrelated to test substance administration.
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Details on results:
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.


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.

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.

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.

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 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.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Changes in pregnancy duration:
effects observed, non-treatment-related

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Reduction in number of live offspring:
effects observed, non-treatment-related
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Changes in postnatal survival:
effects observed, non-treatment-related
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Small decreases in mean spleen weight parameters were observed in the F1 and F2 male and female weanlings (9% - 15%). These decreases were interpreted to be test substance-related.
Details on embryotoxic / teratogenic effects:
There were no reproductive effects or toxicity to F1 and F2 offspring at concentrations up to 1500 ppm, the highest concentration tested.

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased spleen weight in F1 and F2 weanlings. No reproductive toxicity was seen at any concentration.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive toxicity was seen at any concentration. Effects were seen in F1 and F2 weanlings.

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

F1 Males:

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.

Organ Weight Data:

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 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 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 Females:

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.  Therefore, the apparent delay in vaginal opening was not considered test substance-related.

Organ Weight Data:

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 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 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.

F2 Males:

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 F2 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:

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 F2 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.

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.

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:
The existing toxicology data package supports the conclusion that copper has no developmental toxicity potential.
Executive summary:

With the addition of Mylchreest, 2005 to the existing toxicology data base, it is considered that sufficient information is available to adequately evaluate the developmental toxicity potential of copper. This study is particularly relevant, as the rat is considered the best animal model for evaluating the potential hazard effects on human populations.

The 2 generation oral reproduction study carried out by Mylchreest (2005), performed in accordance with OECD test guideline 416, provides information on the effects of repeated exposure to the test substance during all phases of the reproductive cycle including gestation. In particular, the study provides information on the reproductive parameters, and on development, growth and survival of offspring.

Notably, investigation of F1 and F2 litters showed no test substance related effects on the following parameters:

  • pups survival, sex ratio, and survival indices during the lactation period, body weights and clinical observations during lactation,
  • macroscopic examination of pups that died during the lactation period, period of weanings with external abnormalities or clinical signs and of randomly selected weanings,
  • microscopic observations of any gross findings and of liver and brain from randomly selected high-dose and control weanlings.

It is therefore considered that all major manifestations of developmental toxicity (including mortality, structural abnormality, altered growth and functional deficiency) are adequately investigated in this study.

The existing toxicology data package supports the conclusion that copper has no developmental toxicity potential.