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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-02-19 to 2003-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

The purposes of this study were to provide a preliminary evaluation of the potential maternal and developmental toxicity of copper hydroxide when administered orally, by gavage, to pregnant rabbits during the period of major organogenesis, gestation days 7-28, and to provide information for dose level selection for a definitive developmental toxicity study.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Hra:(NZW)SPF rabbit
- Source: COVANCE, Denver, Pennsylvania
- Age at gestation day 0: approx. 5 to 6.5 months old
- Weight at gestation day 0: 2885 to 4330 grams
- Housing: housed individually in suspended, wire-mesh, stainless steel cages
- Diet (approx. 150 g/day): Certified High Fiber Rabbit LabDiet® 5325 (supplier: PMI Nutrition International, Inc.)
- Water (ad libitum)
- Quarantine period: 5 days

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methylcellulose in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- dosing formulations of the test substance in the vehicle were prepared daily.
- bulk test substance was weighed and the vehicle was added to wet the bulk test substance
- additional vehicle was added to achieve the targeted concentration.
- formulations were suspended by continuous stirring for at least 15 minutes prior to the initiation of dosing; additionally, formulations were stirred continuously during dosing.
- dosing volume: 1 mL/kg
- volume administered was based on the most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing suspensions samples containing copper at the concentrations of 0, 7.5, 15, and 30 mg/mL were collected. These samples were analyzed to determine homogeneity, concentration, and 5-hour room temperature stability. Dosing suspensions from all levels were collected and analyzed for uniformity of mixing and concentration verification.

All dosing suspension samples were collected on the same day the suspensions were prepared. They were analyzed when received or stored frozen until analyzed.

The suspension vehicle was aqueous 0.5% methylcellulose.

Analytical methods:
- Dosing suspension treatment:
Each dosing sample (3 mL) was diluted in a 100 mL volumetric with approximately 50 mL of Nanopure® water followed by the addition of 5 mL of nitric acid (69.0%-70.0% pure). This was mixed to dissolve the copper then brought to volume with Nanopure®ID water. The samples were further diluted with Nanopure® water to an expected concentration of approximately 0.001125 mg/mL (a.i.) prior to analysis. Before all final dilutions of the samples, 5 mL of nitric acid (69.0%-70.0% pure) was added along with the 0 mg/mL sample (initial dilution) to each aliquot to give an equivalent final concentration of the matrix (diluted methylcellulose/nitric acid) in all samples.

ICP-AES conditions:
Instrument: Leeman Labs, Inc., ICP-AES, Model PS 1000UV
Power: 1.0 Kw
Coolant: 18 LPM
Nebulizer: 40 psi
Auxiliary: Variable LPM (0.0 - 2.0)
Pump Rate: 1.2 mL/min.

Calibration and quantitation:
An analytical reference standard of 100 ppm stock solution of copper was purchased. Before analysis, appropriate aliquots of the stock were diluted with the matrix (diluted methylcellulose/nitric acid) for the samples and 5% nitric acid in Nanopure® water to make calibration standards, which bracketed the target concentration of the diluted dosing samples. The intensity measurements from the ICP-AES analysis of these standards were used to construct a calibration curve by linear regression (weighted, quadratic). Measured concentrations for dosing suspensions were determined by applying the intensity measurements from the ICPAES for replicate analyses of each sample to the calibration curve.

Test substance homogeneity/uniformity in the vehicle was evaluated by calculating the coefficient of variation (C.V. =standard deviation/mean x 100) ofthe measured concentrations in the top, middle, and bottom samples (homogeneity) or duplicate samples (concentration verification) for each dosing level. A coefficient of variation of less than or equal to 10% is considered indicative of acceptable distribution of the test substance throughout the formulation.

The mean result of the homogeneity samples or concentration verification duplicate samples for each dosing level was used to determine the concentration of the test substance for the respective dosing levels.

Stability was evaluated by using the mean result of the homogeneity samples as the baseline for comparing the corresponding stability results.

Results:
- Homogeneity and stability samples:
The data for samples collected indicates that the test substance was homogeneously mixed in the vehicle at all levels (CV's = 3, 1, 2%, respectively). The test substance was at the targeted concentration in the samples(± 6.0% of nominal) and was stable in the vehicle when held 5 hours at room temperature.
Test substance was not found in the 0 mg/mL samples.

- Concentration verification samples:
The data for samples collected indicate that the test substance was uniformly suspended in the vehicle at all levels (C.V.'s = 3%, 2%, and 2%, respectively) and the test substance was at the targeted concentration in the samples (± 4.0% of nominal).

- Analytical conclusions:
Data from the analysis of the homogeneity, concentration, and stability samples indicate that the test substance was mixed uniformly, was at the targeted levels, and was stable under the conditions study. Test substance was not found in the 0 mg/mL samples.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (upon receipt by the laboratory the does were requested to be at 1 or 3 days of gestation)

Duration of treatment / exposure:

Days 7 - 28 of gestation

Frequency of treatment:

once daily

Duration of test:

29 days

Dose / conc.:

7.5 mg/kg bw/day (actual dose received)

Remarks:

based on copper

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

based on copper

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

based on copper

No. of animals per sex per dose:

Control group: 5 pregnant females
7.5 mg/kg/day: 8 pregnant females
15 mg/kg/day: 9 pregnant females
30 mg/kg/day: 8 pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels for this pilot study were based on the results of a 23-day tolerability study of copper hydroxide in non-pregnant rabbits (DuPont Haskell Laboratory, 2003)*. In the tolerability study, groups of five nonpregnant Hra:(NZW)SPF rabbits were dosed once daily by oral gavage at dose levels of 0, 7.5, 15, or 30 mg/kg/day; all doses were adjusted for copper content.

Results:
15 and 30 mg/kg/day: evidence of toxicity was observed.

30 mg/kg/day:
- 2/5 rabbits were found dead after one or two doses.
- the three surviving rabbits from this group exhibited reduced food consumption and body weight gains during the first week of dosing. However, the survivors appeared to recover after the first week; they resumed eating and began gaining weight.

15 mg/kg/day:
- food consumption and body weight gains were reduced during the first few days of dosing; after these initial reductions, these animals resumed eating and began gaining weight.

7.5 mg/kg/day: data for animals dosed at 7.5 mg/kg/day were generally comparable to the control group
- no-observed-effect level (NOEL) was considered to be 7.5 mg/kg/day was considered the no-observed-effect level (NOEL).

The same dose levels were selected for the current study to permit comparison of toxicity between pregnant and non-pregnant rabbits relative to maximum tolerated maternal dose level and to enable dose level selection for the subsequent definitive developmental toxicity study (please refer to Section 7.8.2 Developmental toxicity / teratogenicity: k_Munley_2003). Despite the fact that 30 mg/kg/day produced mortality in the tolerability study, it was deemed a reasonable high dose level for this study based upon the recovery of the surviving animals. The intermediate level of 15 mg/kg/day was expected to produce minimal maternal toxicity and the low dose level of7.5 mg/kg/day was expected to produce minimal to no maternal toxicity.

*Reference:
- DuPont Haskell Laboratory. (2003). Copper: A 23-Day Tolerability Study in Non-Pregnant Rabbits. Unpublished Report, DuPont Haskell Laboratory Report No. DuPont-11762.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on gestation days 4 - 29, during the dosing period clinical signs were recorded in the morning prior to dosing and once again in the afternoon of each dosing day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily on gestation days 4 - 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 29
Does were euthanized by an intravenous injection of sodium pentobarbital.
A gross external and visceral examination was performed immediately after euthanasia. Sections of adult livers and kidneys,
and spleens were retained at necropsy for possible future histopathological evaluation.Lesions noted were retained for further examination at the discretion.

- animals found dead, sacrificed in extremis, or accidentally killed: gross external and visceral examination was performed within 24 hours.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, females surviving to scheduled euthanasia only
Examinations included:
- Gravid uterus weight: Yes, intact and empty uterus of each doe having at least one viable foetus was weighed to permit calculation of maternal body weight adjusted to exclude the products of conception.
- Number of corpora lutea: Yes, for each ovary of does with viable fetuses
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead and live foetuses: Yes
- Intrauterine location of each foetus was recorded
- uterus of each apparently "nonpregnant" doe was stained with ammonium sulfide to detect very early resorptions, counts were recorded

- animals found dead, sacrificed in extremis, or accidentally killed: pregnancy status was determined by the presence or absence of implantation sites; uterinecontents were described to the extent possible.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Foetuses were euthanized by an intraperitoneal injection of sodium pentobarbital after external examination was complete.
- Foetal body weight: Yes, each foetus
- For two does, one each at 7.5 and 30 mg/kg/day, foetal livers were weighed and retained for possible future histopathological evaluation.

Statistics:

Descriptive statistics (mean, standard deviation) were performed for all maternal and foetal quantitative data.

Indices:

not specified

Historical control data:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

7.5, 15, and 30 mg/kg/dayy:
- low incidences of diarrhea were observed at all dose levels (considered compound-related).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

30 mg/kg/day:
- compound-related mortality was produced.
- one rabbit was sacrificed in extremis on gestation day 9 and another rabbit was found dead on gestation day 26.

Gross necropsy findings indicated stomach hemorrhages for animal 35872 and a small liver that was moderately autolyzed for animal 35859. For animal 35872, selected tissues were retained and examined microscopically; the summary of pathology findings indicated that the cause of death was related to a hemolytic event that resulted in hemoglobin nephropathy and probably renal failure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

15 and 30 mg/kg/day:
- clear compound-related reductions in maternal body weight and weight change were produced. In general, decrements in these parameters were marked and dose-related during the early part of the dosing period. After about a week, the animals typically recovered and resumed gaining weight. However, the magnitude of the reductions early on was sufficiently marked such that the overall impact of the reductions was persistent.

30 mg/kg/day:
- maternal body weight losses were pronounced during the early part of the dosing period; overall body weight gain during the dosing period was reduced by 88% relative to the control group.
- final body weights were 9% lower than the controls as a result of the decrements in weight gain and food consumption.

15 mg/kg/day:
- overall weight gain means was 11% lower than the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

15 and 30 mg/kg/day:
- clear compound-related reductions in maternal food consumption were produced. In general, decrements in the parameter was marked and dose-related during the early part of the dosing period. After about a week, the animals typically recovered and resumed eating. However, the magnitude of the reductions early on was sufficiently marked such that the overall impact of the reductions was persistent.

30 mg/kg/day:
- food consumption reductions were marked and persistent; overall food consumption during the dosing period was 44% lower than the control group.

15 mg/kg/day:
- overall food consumption means was 22% lower than the control group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

30 mg/kg/day:
- animal sacrificed in in extremis: gross necropsy findings indicated stomach hemorrhages For this animal selected tissues were retained and examined microscopically; the summary of pathology findings indicated that the cause of death was related to a hemolytic event that resulted in hemoglobin nephropathy and probably renal failure.
- animal found dead: gross necropsy findings indicated a small liver that was moderately autolyzed.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS:
- 7.5, 15, and 30 mg/kg/day: other clinical observations (alopecia, gasping, lung noise, and stained tail) were either incidental, infrequent, and/or not dose-related.

MORTALITY:
7.5, 15, and 30 mg/kg/day:
- five animals (two each from the 7.5 and 15 mg/kg/day groups and one from the 30 mg/kg/day group) were either accidentally killed or were found dead as the result of intubation injuries (not considered compound-related).

BODY WEIGHT:
- 7.5 mg/kg/day. no compound-related effects on maternal body weight.

FOOD CONSUMPTION:
- 7.5 mg/kg/day. no compound-related effects on maternal food consumption.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

30 mg/kg/day:
- foetal resorptions appeared slightly increased at this level

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Other effects:

not examined

Remarks on result:

other:

Remarks:

No compound-related maternal toxicity was observed at 7.5 mg/kg/day. Maternal toxicity at 15 and 30 mg/kg/day included dose-related reductions in body weight and food consumption parameters. The evidence of toxicity produced at 30 mg/kg/day was marked and included maternal mortality; postmortem evaluation of the does at 30 mg/kg/day revealed findings including gastric ulcers, evidence of hemolytic anemia, and renal damage. Thus, 30 mg/kg/day was not tolerated in pregnant rabbits.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

30 mg/kg/day:
- mean foetal weights were reduced by 12% relative to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

30 mg/kg/day:
- 4 foetuses (2 each from 2 litters) were observed with omphalocele (protrusion of intestines at the umbilicus).

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

7.5 and 15 mg/kg/day
- no evidence of developmental toxicity.
- one foetus at 7.5 mg/kg/day had anasarca, domed head, and a short tail (not considered compound-related)

Remarks on result:

other:

Remarks:

No compound-related developmental toxicity was observed at 7.5 or 15 mg/kg/day. Developmental toxicity at 30 mg/kg/day included reduced mean fetal weight, slightly increased resorptions, and increased malformations (omphalocele was observed in four fetuses from two litters).

Conclusions:

Under the conditions of the current study, no compound-related maternal toxicity was observed at 7.5 mg/kg/day and no compound-related developmental toxicity was observed at 7.5 or 15 mg/kg/day. Maternal toxicity at 15 and 30 mg/kg/day included dose-related reductions in body weight and food consumption parameters. The evidence of toxicity produced at 30 mg/kg/day was marked and included maternal mortality; postmortem evaluation of the does at 30 mg/kg/day revealed findings including gastric ulcers, evidence of hemolytic anemia, and renal damage consistent with literature reports of acute copper toxicity in other species. Thus, 30 mg/kg/day was not tolerated in pregnant rabbits. Developmental toxicity at 30 mg/kg/day included reduced mean fetal weight, slightly increased resorptions, and increased malformations (omphalocele was observed in four fetuses from two litters).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1987

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Copper dihydroxide

EC Number:

243-815-9

EC Name:

Copper dihydroxide

Cas Number:

20427-59-2

IUPAC Name:

copper(2+) dihydroxide

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): copper hyroxide
- State of aggregation: free flowing blue product; needles
- Particle size (average): 4.6 µm
- Moisture content: 2.27 %

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS - Hra:(NZW)SPF rabbit
- Source: COVANCE, Denver, Pennsylvania
- Age at gestation day 0: approx. 5 months old
- Weight at gestation day 0: 2988 to 4412 grams
- Housing: housed individually in suspended, wire-mesh, stainless steel cages
- Diet (approx. 150 g/day): Certified High Fiber Rabbit LabDiet® 5325 (supplier: PMI Nutrition International, Inc.)
- Water (ad libitum)
- Quarantine period: at least 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous methylcellulose in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- dosing formulations of the test substance in the vehicle were prepared daily.
- bulk test substance was weighed and the vehicle was added to wet the bulk test substance
- additional vehicle was added to achieve the targeted concentration.
- formulations were suspended by continuous stirring for at least 30 minutes prior to the initiation of dosing; additionally, formulations were stirred continuously during dosing.
- dosing volume: 1 mL/kg
- volume administered was based on the most recent body weight.
- after each dose was delivered, a small quantity of vehicle was used to flush the gavage catheter prior to removal of the catheter from the stomach. The purpose of this post-dose flush was to clear the tube of the dosing formulation and thereby prevent any residual formulation from being deposited in the mouth upon removal where it could either be aspirated or induce a taste aversion response in the rabbits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations samples containing the copper at the concentrations of 0, 6, 9, and 18 mg/mL were collected on test day 1. These samples were analyzed to verify homogeneity, concentration, and 5-hour room-temperature stability. Samples from all concentrations were collected on test day 10 and test day 22 and analyzed to verify uniformity of mixing and concentration. All samples were collected on the same day the formulations were prepared.

Analytical methods:
1) Recovery sample analysis:
- recovery of copper from spiked 0.5 % aqueous methylcellulose was tested at 6, 9, and 18 mg/mL levels to confirm the analytical method.
- measured amounts of copper hydroxide were weighed (approximately 30, 45, and 90 mg for the evels, respectively) and diluted with 0.5% aqueous methylcellulose.
- all recovery samples were mixed to disperse the copper hydroxide in the methylcellulose.
- samples were processed and analyzed in the same manner as the dosing samples at similar concentrations.

2) Dosing formulation sample treatment
- each dosing sample was diluted with Nanopure® water followed by the addition of nitric acid (69.0-70.0% pure). This was mixed to dissolve the copper then brought to volume with Nanopure® water.
- samples were further diluted with Nanopure® water to an expected concentration of approximately 0.00108 mg/mL (a.i.) prior to analysis.
- before all final dilutions of the samples, nitric acid (69.0-70.0% pure) was added along with the 0 mg/mL sample (initial dilution) to each aliquot to give an equivalent final concentration of the matrix (diluted methylcellulose/nitric acid) in all samples.

3) ICP-AES conditions
Instrument: Leeman Labs, Inc., ICP-AES, Model PS 1000UV
Power: 1.0 Kw
Coolant: 18 LPM
Nebulizer: 40 psi
Auxiliary: Variable LPM (0.0 - 2.0)
Pump Rate: 1.2 mL/min.

4) Calibration and quantitation:
- an analytical reference standard of 100 ppm stock solution of copper was obtained.
- before analysis, appropriate aliquots of the stock were diluted with the matrix (diluted methylcellulose/nitric acid) for the samples and 5% nitric acid in Nanopure® water to make calibration standards, which bracketed the target concentration of the diluted dosing samples.
- the intensity measurements from the ICP-AES analysis of these standards were used to construct a calibration curve by linear regression (weighted, quadratic).
- measured concentrations for dosing formulations were determined by applying the intensity measurements from the ICP-AES for replicate analyses of
each sample to the calibration curve.
- test substance homogeneity and uniformity in the vehicle was evaluated by calculating the coefficient of variation of the measured concentrations in the top, middle, and bottom samples (homogeneity) or duplicate samples (concentration verification) for each dosing level.
- a coefficient of variation of less than or equal to 10 % is considered indicative of acceptable distribution of the test substance throughout the solution.
- the mean result of the homogeneity samples or concentration verification duplicate samples for each dosing level was used to determine the concentration of the test substance for the respective dosing levels.
- stability was evaluated by using the mean result of the homogeneity samples as the baseline for comparing the corresponding stability results.

Results:
1) Recovery samples:
Measured concentrations of copper:
- 6 mg Cu/mL level: 99.7 % to 103.2 % of nominal (mean percent recovery= 101.0 % ± 1.9, C.V. = 2 %).
- 9 mg Cu/mL level: 97.6 % to 114.7 % of nominal (mean percent recovery = 103.8 % ± 9.4, C.V. = 9 %)
- 18 mg Cu/mL level: 99.4 % to 100.0 % of nominal (mean percent recovery= 99.6 % ± 3.2, C.V. = 3 %).
Based on this data, the analytical method performed satisfactorily over the entire concentration range of the study.

2) Homogeneity and stability samples:
The data for samples collected on test day 1 indicates that the test substance was homogeneously mixed in the vehicle at all levels (CV's = 3 %, 2 %, 3 %, respectively). The test substance was a tthe targeted concentration in the samples (± 2.8 % of nominal) and was stable in the vehicle when held 5 hours at room temperature.
Test substance was not found in the 0 mg/mL samples.

3) Concentration verification samples:
The data for samples collected on test day 10 indicate that the test substance was uniformly suspended in the vehicle at all levels (C.V.'s = 0.7%, 2%, and 7%, respectively) and the test substance was at the targeted concentration in the samples(± 9.2% of nominal).
The data for samples collected on test day 22 indicate that the test substance was uniformly suspended in the vehicle at all levels (C.V.'s = 2 %, 0.04 %, and 2 %, respectively) and the test substance was at the targeted concentration in the samples(± 4.2 % of nominal).

5) Conclusions:
Data from the analysis of the homogeneity, concentration verification, and stability samples indicate that the test substance was mixed uniformly, was at the targeted levels, and was stable under the conditions of the study. The data for the concentration verification samples indicated that the test substance was mixed uniformly in the vehicle and at the targeted concentrations during the study. Test substance was not found in the 0 mg/mL samples.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (upon receipt by the laboratory the does were at either 1 or 2 days of gestation)

Duration of treatment / exposure:

Days 7 - 28 of gestation

Frequency of treatment:

once daily

Duration of test:

29 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Remarks:

based on copper

Doses / concentrations 2

Dose / conc.:

9 mg/kg bw/day (actual dose received)

Remarks:

based on copper

Doses / concentrations 3

Dose / conc.:

18 mg/kg bw/day (actual dose received)

Remarks:

based on copper

No. of animals per sex per dose:

22 pregnant females (exception: 21 pregnant females in the 18 mg Cu/kg/day dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this current study were based on results of a pilot study conducted with the test substance (please refer to Section 7.8.2 Developmental toxicity / teratogenicity: k_Munley_2003)*. In the pilot study, suspensions of copper hydroxide in 0.5% aqueous methylcellulose in deionized water were administered once daily by gavage to groups of 5, 8, 9, and 8 time-mated Hra:(NZW)SPF rabbits over days 7-28 of gestation at dose levels of 0, 7.5, 15, and 30 mg Cu/kg/day, respectively. The daily doses were administered at a volume of 1 mL/kg. Clinical observations, body weights, and food consumption were recorded daily on gestation days 4 - 29. On gestation day 29, surviving females were euthanized and examined grossly (external and visceral examination). The uterus of each pregnant female was removed and the uterine contents were examined and described; all foetuses were individually identified, weighed, sexed, and examined for external alterations.
For females that were found dead, sacrificed in extremis, or accidentally killed, a gross external and visceral examination was performed within 24 hours. Pregnancy status was determined by the presence or absence of implantation sites; uterine contents were described to the extent possible.

Results:
0 mg Cu/kg/day:
- no external malformations were observed in foetuses.

7.5 mg Cu/kg/day:
- no test substance-related maternal toxicity was observed.
- no evidence of test-substance related developmental toxicity.
- foetal malformations observed was anasarca, domed head, and a short tail (not compound-related finding).

15 mg Cu/kg/day:
- maternal toxicity included dose-related reductions in body weight and food consumption parameters
- no evidence of test-substance related developmental toxicity.
- no external malformations were observed in foetuses.

30 mg Cu/kg/day:
- maternal toxicity included dose-related reductions in body weight and food consumption parameters (marked at 30 mg/kg/day);
- compound-related maternal mortality was observed (2 rabbits)(postmortem evaluation: gastric ulcers, evidence of hemolytic anemia, and renal damage).
- evidence of compound-rrelated developmental toxicity: reduced mean foetal body weight and a slightly increased incidence of foetal resorptions.
- foetal malformations observed was omphalocele.

The results of the pilot study suggest that the foetus is not uniquely sensitive to test substance-related toxicity, as effects on the foetus were only evident at a level that produced evidence of marked maternal toxicity including mortality. Based on these results, the doses for the main study were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on days 4 - 29 of gestation (remark: during the dosing period (days 7-28 of gestation), clinical signs were recorded in the morning prior to dosing and once again in the afternoon of each dosing day.)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily on days 4 - 29 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
Females were euthanized by an intravenous injection of sodium pentobarbital.
A gross external and visceral examination was performed immediately after euthanasia. Lesions noted were retained for further examination at the discretion.

- females found dead or accidentally killed: gross external and visceral examination was performed within 24 hours.
- females that aborted or delivered before scheduled euthanasia: they were euthanized and a gross external and visceral examination was performed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes, females surviving to scheduled euthanasia only
Examinations included:
- Gravid uterus weight: Yes, intact and empty uterus of each female having at least one viable foetus was weighed to permit calculation of maternal body weight adjusted to exclude the products of conception.
- Number of corpora lutea: Yes, for each ovary of females with viable fetuses
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead and live foetuses: Yes
- Position of implants was recorded
- Intrauterine location of each foetus was recorded
- uterus of each apparently "nonpregnant" female was stained with ammonium sulfide to detect very early resorptions.

- females found dead or accidentally killed: determination of presence or absence of implantation sites. uterine contents were described to the extent possible. Uterus of each apparently non-pregnant female was stained with ammonium sulfide to detect very early resorptions. Lesions and/or grossly abnormal uterine contents were recorded by exception as a maternal gross postmortem finding and were retained for further examination at discretion.

- females that aborted or delivered before scheduled euthanasia: lesions and/or grossly abnormal uterine contents were recorded by exception as a maternal gross postmortem finding and were retained for further examination at the discretion.

Fetal examinations:

- Females that aborted or delivered before scheduled euthanasia: any foetuses delivered or those remaining in utero were discarded without foetal evaluation.

- Foetuses were euthanized by an intraperitoneal injection of sodium pentobarbital after external examination was complete.

- Foetal body weight: Yes, each foetus

All live foetuese were examined as follows:
- External examinations: Yes
- Soft tissue examinations: Yes
Retarded renal development was classified using the scheme of Woo and Hoar (1972)*.
- Skeletal examinations: Yes
- Head examinations: Yes, brain and eyes

- Foetal sex of alive foetuses: Yes

*Referenz:
- Woo, D. C. and Hoar, R. M. (1972). Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats: The effect of Methyl Salicylate. Teratology. 6:191-196.

Statistics:

Descriptive statistics were calculated for each quantitative parameter listed below. Sequential two-tailed trend testing (Selwyn, 1995)* was applied to the data for each parameter as tabulated below. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. For litter parameters, the proportion of affected fetuses per litter or the litter mean was used as the experimental unit for statistical evaluation (Haseman & Hogan, 1975)*.
The level of significance selected was p < 0.05. Where the data were tied and the standard large sample version of Jonckheere's test was not applicable, exact p values were calculated using permutation methodology (Patefield, 1982)*.

Parameters and trend tests:
- Linear contrast of means (Snedecor & cochran, 1967)*: maternal weight, maternal weight change, and maternal food consumption
- Lonckheere's test (Jonckheere, 1954)*: live foetuses, dead foetuses, resorptions, implantations, and incidence of foetal alterations
- Cochran-Armitage test (Snedecor & cochran, 1967)*: incidence of pregnancy, clinical observations, maternal mortality, females with total resorptions, and abortions/early deliveries
- linear contrast of least square means (Dempster et al., 1984)*: foetal weight (Covariates: litter size, sex ratio) and sex ratio (Covariate: litter size)

* References:
- Selwyn, M.R. (1995). Journal of the American College of Toxicology. 14(2): 158-168.
- Haseman, J. K. and Hogan, M.D. (1975). Teratology. 12: 165-171.
- Patefield, W. (1982). Applied Statistics. 31: 32-43.
- Snedecor, G.W. and Cochran, W. G. (1967). Statistical Methods, 6th edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa.
- Jonckheere, A. R. (1954). Biometrika. 41, 133-145.
- Dempster, A. P. et al. (1984). The Journal of the Royal Statistical Society. Series C (Applied Statistics). 33(2): 203-214.

Indices:

not specified

Historical control data:

not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

18 mg Cu/kg/day:
- test substance-related clinical observations occurred (significantly increased incidence of diarrhea (9/21 females); stained fur(tail) (5/21 females); cageboard staining (3/21 females); irregular respiration (1/21 females); weakness (1/21 females))
- diarrhea was relatively transient and was evident on single days or intermittently over several days.
- stained fur and cageboard were related to the diarrhea and/or abortions.

9 mg Cu/kg/day:
- test substance-related clinical observations occurred (significantly increased incidence of diarrhea (5/22 females))
- diarrhea was relatively transient and was evident on single days or intermittently over several days.

6 mg Cu/kg/day:
- test substance-related clinical observations occurred (significantly increased incidence of diarrhea (5/21 females))
- diarrhea was relatively transient and was evident on single days or intermittently over several days.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

18 mg Cu/kg/day:
- test substance-related mortality was observed
- 3 rabbits were found dead between gestation days 9 and 16

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

9 and 18 mg Cu/kg/day:
- test substance-related, statistically significant, adverse effects on maternal body weights and weight changes
These reductions were primarily the result of marked, significant, dose-related body weight losses that occurred over the first three days of dosing (gestation days 7 - 10). The weight losses and/or reduced gains tapered off by the end of the first week of dosing with the subsequent gains for these groups being comparable to the control group. However, the effect at the beginning of the dosing period was sufficiently pronounced such that overall body weight gain at 9 and 18 mg Cu/kg/day was adversely impacted; overall gains (gestation days 7-29) were 31% and 72% lower than for the control group at 9 and 18 mg Cu/kg/day, respectively. As a result, mean body weights at 9 and 18 mg Cu/kg/day were lower or significantly lower beginning on gestation day 10 and persisting to some extent until near the end of dosing.

6 mg Cu/kg/day:
- test substance-related effects on maternal body weight.
These effects consisted of slight and/or occasionally significant reductions in body weight gain at the beginning of the dosing period (gestation days 7-10). Overall weight gain (gestation days 7-29) was 20% lower than for the control group, respectively; however, this reduction was not significant and had no
impact on final body weights which were comparable to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

9 and 18 mg Cu/kg/day:
- there were marked, statistically significant reductions in mean food consumption during the first week of dosing (gestation days 7 - 10 and 10 -13). At 18 mg Cu/kg/day, mean food consumption remained reduced until the end of the study. Overall food consumed was significant and was 17 % and 30 % lower than for the control group at 9 and 18 mg Cu/kg/day, respectively.

6 mg Cu/kg/day:
- test substance-related effects on maternal food consumption.
These effects consisted of slight and/or occasionally significant reductions in food consumption at the beginning of the dosing period (gestation days 7-10). Overall food consumption (gestation days 7-29) was 6% lower than for the control group, respectively; however, this reduction was not significant and had no
impact on final body weights which were comparable to the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

18 mg Cu/kg/day
- 3 animals (died prior to scheduled euthanasia): stomach hemorrhage and/or ulceration, dark discolouration or mottling of lung tissue, pale liver, gelatinous tan rectal discharge, and brown liquid in chest cavity.
- 1 animal (aborted): red discoloured stomach lining at necropsy.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Mortality:
- 18 mg Cu/kg/day: 1 animal was accidentally killed (intubation injury) on gestation day 15.
- 9 mg Cu/kg/day: no test substance-related mortality was observed.
- 6 mg Cu/kg/day: no test substance-related mortality was observed.

Gross pathology:
18 mg Cu/kg/day
- 1 animal (accidental kill): stomach hemorrhage and evidence of gavage injury to lung tissue.

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

18 mg Cu/kg/day:
- two animals aborted on gestation day 22 and were subsequently euthanized.
- aborted materials for these two animals consisted primarily of placentae and apparently resorbing foetuses.

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

Number of abortions:
6 mg Cu/kg/day: 1 animal aborted on gestation day 27 and was subsequently euthanized (not considered test substance-related; not clearly dose-related; within the range of historical control data).

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

6 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

copper

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: number of abortions

Description (incidence and severity):

18 mg Cu/kg/day:
- two animals aborted on gestation day 22
- aborted materials for these two animals consisted primarily of placentae and apparently resorbing foetuses.

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

18 mg Cu/kg/day:
- 9 % reduction in mean foetal weight
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

18 mg Cu/kg/day:
- significant increases in the mean percent affected foetuses per litter with hemivertebrae (2/126 foetuses), supernumerary ribs (110/126 foetuses), and delayed skull ossification (5/126 foetuses)

Visceral malformations:

no effects observed

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

copper

Sex:

male/female

Basis for effect level:

other: mean foetal body weight and skeletal variations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: rib

skeletal: vertebra

Description (incidence and severity):

18 mg Cu/kg/day:
- significant increases in the mean percent affected foetuses per litter with hemivertebrae (2/126 foetuses), supernumerary ribs (110/126 foetuses), and delayed skull ossification (5/126 foetuses)

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

18 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:

NOAEL (maternal toxicity): 6.0 mg Cu/kg/day
NOEL (developmental toxicity): 9.0 mg Cu/kg/day

Under the conditions of the current study, there was no evidence of test substance-related teratogenicity. The maternal no-observed-adverse-effect level (NOAEL) was 6 mg Cu/kg/day based on reduced body weight gain and food consumption observed at 9 mg Cu/kg/day and overt toxicity including maternal mortality at 18 mg Cu/kg/day. The no-observed-effect level (NOEL) for foetuses was 9 mg Cu/kg/day based on a slight reduction in mean foetal body weight and a slight increase in minor skeletal variations observed at 18 mg Cu/kg/day.