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Administrative data

Description of key information

One well performed and reported subchronic oral toxicity study was available.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January- April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study under GLP
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: (RccHan™:WIST) were obtained from a colony maintained under SPF-conditions at Harlan, the Netherlands
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 166 - 209 g (mean 187 g) for males and from 130 – 177 g (mean 145 g) for females
- Fasting period before study: not applicable
- Housing: 4 per sex in macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) and a wooden block as environmental enrichment.
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-70 except during a few brief periods generally associated with room cleaning. The maximum value recorded during these short periods was 98%. On one occasion (6 February, 2012), the relative humidity dropped below 45% during about one hour, reaching a minimum value of 37%.
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 January To: 20 April 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dilutions of the test substance in the vehicle were prepared weekly and used within 7 days after preparation. For each dose level, the dilutions of the test substance in tap water were prepared by adding weighed amount of test substance to tap water and stirring on a magnetic stirrer until a homogeneous solution was obtained. After making up the total volume to obtain the appropriate concentration (w/v), 8 aliquots (7 days plus 1 extra as reserve) per dose level were taken according to the daily volume required for each dosing.

VEHICLE: tap water
- Concentration in vehicle: 0, 15, 50 and 150 mg/mL. Due to mortality, the high dose was lowered to 10.5 mg/L from day 9 onwards.
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken from each dosing formulations prepared in the study. Analyses to determine the content and homogeneity of the test substance in the carrier were conducted in the first batch used in the study, by analysing copper with Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) in three samples (taken at the top, mid and bottom of the container) per dose level (15, 50 and 150 mg/ml).
In addition, the content of the test substance was determined in two other batches of dosing dilutions used in the study level (15, 50 and/or 105 mg/ml) by analysing one sample per concentration level. Because the test substance is known to be stable in the carrier, analyses for stability of the test substance in the dosing dilutions were not conducted.
Duration of treatment / exposure:
Male animals were dosed during a 10-week premating period, during mating and up to the day before scheduled sacrifice (day 90).
Surviving female animals were dosed with the test substance during a 10-week premating period, and during mating, gestation and lactation up to the day before scheduled sacrifice (day 4 of lactation). Animals in moribund condition were dosed until they died or were humanely killed. In a few cases, the dosing was interrupted on the day of death or one day before (rat no’s 73, 82, 83, 86, 92, 94).
The dosing volume was 10 ml/kg body weight. Dose volume was adjusted to the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volume was not adjusted after GD 14.
Frequency of treatment:
single daily application by gavage
Remarks:
Doses / Concentrations:
0, 150, 500 and 1500/1050 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on studies done with EDTA and EDTA-MnNa2
- Rationale for animal assignment (if not random): computer randomization proportionately to BW
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observations outside the home cage were made once weekly (10 rats/sex/group)

NEUROBEHAVIOURAL EXAMINATION: Yes (10 rats/sex/group)
- Time schedule for examinations: in week 9 (females) or week 10 (males) of the pre-mating period
- Dose groups that were examined: all
- Battery of functions tested: FOB (including sensory activity and grip strength) and spontaneous motor activity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (males and females) and on day 1 and 4 of lactation (females) and prior to scheduled necropsy

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: weekly (at same time as measurement of bw)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic observations were made prior to the start of the treatment (on day -7[♂] or day -8[♀]) in all rats of all groups, and at the end of thepremating period in all rats of the control group and the mid-dose group and in the remaining rats of the high-dose group (on day 62[♀] or day 64[♂]).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in females on day 65, a few days prior to the start of the mating period (day 70), and in males on day 85, a few days prior to their necropsy (day 90)
- Anaesthetic used for blood collection: Yes Blood was collected by orbita punction under CO2/O2 anaesthesia
- Animals fasted: Yes (water freely available)
- How many animals: 10 sex/group
- Parameters checked: haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in females on day 65, a few days prior to the start of the mating period (day 70), and in males on day 85, a few days prior to their necropsy (day 90)
- Animals fasted: Yes (water freely available)
- How many animals: 10 sex/group
- Parameters checked: alkaline phosphatase activity (ALP), bilirubin (total), aspartate aminotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin (calculated), potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate (PO4), glucose (fasting)

URINALYSIS: No

Sacrifice and pathology:
SACRIFICE
Male animals were sacrificed after 90-days of treatment. Female animals were sacrificed at day 4 of lactation (almost 14 weeks of treatment).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Necropsy was also performed on animals that died intercurrently or that were killed in moribund condition

ORGAN WEIGHTS:
adrenals, ovaries, brain, prostate, epididymides, seminal vesicles with coagulating glands, heart, spleen, kidneys, testes, liver, thymus, uterus.

HISTOPATHOLOGY:
Histopathological examination (by light microscopy) was performed on all tissues and organs listed in OECD 413 of 10 rats/sex of the control and the mid-dose group (those with the lowest identification numbers, and avoiding the not-mated females no’s 13, 61 and 69). And the animals of the control group, the low-dose group and the mid-dose group that died during the study or were killed in extremis (males no’s 52, 58 and 62).
Because of the early mortality in the high-dose group, the rats of this group were not subjected to a complete histopathological examination. Gross lesions were examined histopathologically in all rats of all groups, including those of the high-dose group. Gross lesions in the rats that died in the initial phase of the study and that were replaced by reserve animals were not reported or subjected to microscopic examination. Because treatment-related changes were observed in the kidneys, liver and spleen in mid-dose group, histopathology on these organs was extended to 10 animals/sex of the low-dose groups (those with the lowest identification numbers).

Other examinations:
See at reproduction and developmental toxicity
Statistics:
- Body weight and feed consumption data: one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Clinical pathology (haematology and clinical chemistry) and organ weights: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method (abbreviation AUTO). This test is an automatic decision tree consisting of: (1) Data pre-processing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed, (2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; non-parametric for rank transformed data: Kruskal-Wallis test), (3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Incidences of histopathological changes of parent animals: Fisher’s exact probability test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: All male and female rats of the high-dose group were found dead or killed in moribund condition before the start of the mating period. Three males of the mid-dose group were killed in moribund condition during or at the end of the mating period.
Clinical signs observed during the premating period in rats of the high-dose group and, to a lesser extent, in the mid-dose group included thin appearance, hunched posture, piloerection, blepharospasm, swollen abdomen, soft faeces and green watery discharge. Soft faeces and swollen abdomen were observed in the mid-dose group during gestation. No relevant signs were noted during lactation.

BODY WEIGHT AND FOOD CONSUMPTION: During the premating period, mean body weights were decreased in males of the high-dose group, and, from the end of this period, in males of the mid-dose group. The differences with the controls were statistically significant at most occasions. In females of the high-dose group mean body weights were occasionally higher than in controls. During gestation, there were no significant differences in female body weights between the low- or mid-dose group and the controls. During lactation, body weights were statistically significantly increased in both remaining treatment groups on day 1 and in the mid-dose group on day 4.
Feed intake was statistically significantly decreased in males and females of the high-dose group and in males of the mid-dose group during the first week of the premating period, and remained relatively low in males of the high-dose group during the premating period (the differences with the controls occasionally being statistically significant). Feed intake was not significantly affected in females at other stages of the premating-, gestation- or lactation period, except for an increase in week 6, and a decrease in week 9 in females of the high-dose group.

TEST SUBSTANCE INTAKE: no effects (gavage)

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: no treatment-related effects

HAEMATOLOGY:
- Prothrombin time was decreased in the mid-dose group in both sexes
- Red blood cell count was increased and MCV and MCH decreased in males of the mid-dose group. In females of this group MCHC was decreased.
- Reticulocytes and thrombocytes were increased in the remaining females of the high-dose group.
- Total white blood cell counts and absolute neutrophils and monocytes counts were increased in males of the mid-dose group and in the three remaining females of the high-dose group. The changes in absolute white blood cells were generally reflected in changes in the percentage distribution of differential white blood cells.

CLINICAL CHEMISTRY:
- ALP, ASAT, ALAT and GGT activity were increased in mid-dose males. ASAT activity was also increased in the three remaining high-dose females. However, in females of several groups, ALP activity (mid- and high-dose group) and ALAT activity (low- and mid-dose group) were decreased.
- Bilirubin was increased in males of the mid-dose group and in the three remaining high-dose females.
- Creatinine was increased in the mid-dose group in both sexes and in the three remaining high-dose females.
- Urea, inorganic phosphate and calcium were increased in mid-dose males and in the three remaining high-dose females.
- Electrolytes showed a number of fluctuations; potassium was decreased in low-dose males and increased in mid-dose females, sodium was increased in low- and mid-dose males and chloride was decreased in the three remaining high-dose females.
- Albumin was decreased in the three remaining high-dose females.

URINALYSIS: not measured

NEUROBEHAVIOUR: no treatment-related effects

ORGAN WEIGHTS:
- The absolute and the relative weights of the heart were decreased in the mid-dose group in both sexes.
- The relative weight of the kidneys was increased in males of the mid-dose group. In females of this group, the absolute kidney weight was increased.- The absolute and the relative weights of the spleen were increased in the mid-dose females.
- The absolute and the relative weights of the ovaries were decreased in females of the mid-dose group.
- The absolute weight of the testis was decreased in mid-dose males.
- The absolute and the relative weights of the epididymides were decreased in males of the low-dose group, but this finding was not confirmed by significant changes in the mid-dose group.
- The absolute weight of the thymus was decreased in mid-dose males, and the absolute adrenal weight was increased in mid-dose females, but the relative weights of these organs were not significantly affected.
- The relative weight of the brain was decreased in the mid-dose females. Because the absolute weight of this organ was not affected this finding is ascribed to the higher terminal body weights in females of this group.

GROSS PATHOLOGY: The main gross findings in animals of the mid- and high-dose groups were enlarged intestines with green/watery contents, a pale and/or green appearance of the liver and kidneys, small epididymides and seminal vesicles, enlarged dark spleen, small thymus and a variety of changes in the stomach. These gross changes were ascribed to treatment. A small thymus is most probably secondary to a poor health condition.

HISTOPATHOLOGY (NON-NEOPLASTIC): Microscopic examination of the sampled organs and tissues revealed treatment related histopathological changes in the kidneys, the liver and the spleen.
- The histopathological changes in the kidneys were characterised by tubular necrosis and degeneration, tubular epithelial cell karyomegaly and accumulation of brown pigment.
- The changes in the liver were accumulation of periportal macrophages, especially in the mid-dose males, hepatocellular karyomegaly, brown pigment accumulation, bile duct hyperplasia and (multi)focal infiltration of mononuclear inflammatory cells.
- The changes in the spleen were accumulation of brown pigment and accumulation of macrophages in the white pulp.
The above changes were mainly present in the mid-dose animals. However, tubular epithelial brown pigment was also noted in the kidneys of 6/10 low-dose males and mononuclear cell infiltrate was present in the liver of 6/10 low-dose males and 3/10 low-dose females.
In the high-dose animals, subjected to microscopical examination on the basis of macroscopic observations, most of the above histopathological changes and several more were observed at microscopy. Although these findings are difficult to interpret, because those animals were exposed to the test substance for a shorter time than the animals in the other groups most, if not all, of the histopathological changes observed in the high-dose animals were likely related to treatment.

HISTOPATHOLOGY (NEOPLASTIC): no changes

HISTORICAL CONTROL DATA: not needed

Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL was < 150 mg/kg bw due to limited liver and kidney effects at this level. Based on these limited effects, it is expected that the NOAEL is close to 150 mg/kg bw.
Critical effects observed:
not specified
Conclusions:
Based on the slight effects in liver and kidneys in the low-dose group, the no-observed-effect level (NOEL) for parental toxicity was lower than 150 mg/kg bw/day. However, based on the limited severity of the effects, the NOEL was expected to be close to 150 mg/kg bw.
Executive summary:

In this study, the possible effects of EDTA-CuNa2 on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats. EDTA-CuNa2was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4. The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. Due to mortality, the high-dose level was reduced to 1050 mg/kg bw/day from day 9 of the study.

The content and homogeneity of the test substance in the carrier were confirmed by analysis.

All male and female rats of the high-dose group were found dead or killed in moribund condition before the start of the mating period. Three males of the mid-dose group were killed in moribund condition during or at the end of the mating period.

Clinical signs observed in rats of the high-dose group and, to a lesser extent, in the mid-dose group included thin appearance, hunched posture, piloerection, blepharospasm, swollen abdomen, soft faeces and green watery discharge around perineum.

Neurobehavioural observations and motor activity assessment did not indicate specific neurotoxic effects of the test substance. Ophthalmoscopic examination did not reveal any treatment-related changes.

Body weights were decreased in males of the high-dose group, and, from the end of the premating period, in males of the mid-dose group. During lactation, female body weights were increased in the remaining treatment groups. Feed intake was reduced in males of the high-dose group.

Haematology and clinical chemistry was conductedin females on day 65 (at the end of the premating period), and in males on day 85 (a few days prior to necropsy). At these time points, all males and most females of the high-dose group had died or had been killed.

The following changes in haematology were noted in the mid-dose group (now representing the highest dose level):

-       Prothrombin time was decreased in both sexes.

-       Red blood cell count was increased and MCV and MCH were decreased in males.

-       Total white blood cell counts and absolute neutrophils and monocytes counts were increased in males. 

The following changes in clinical chemistry were noted:

-       ALP, ASAT, ALAT and GGT activity were increased in males of the mid-dose group. In females ALP activity (mid-dose group) and ALAT activity (low- and mid-dose group) were decreased.

-       Bilirubin was increased in males of the mid-dose group

-       Creatinine was increased in the mid-dose group in both sexes

-       Urea, inorganic phosphate and calcium were increased in mid-dose males

-       Sodium was increased in males of the low- and mid-dose group. Potassium was increased in females of the mid-dose group.

The surviving rats (control, low- and mid-dose group) were killed on day 90 (males) or on day 4 of lactation (females).

-       Terminal body weights were decreased in males and increased in females of the mid-dose group.

-       The absolute and the relative weights of the heart were decreased in the mid-dose group in both sexes.

-       The relative weight of the kidneys was increased in males of the mid-dose group. In females of this group, the absolute kidney weight was increased.

-       The absolute and the relative weights of the spleen were increased in the mid-dose females.

-       The absolute and the relative weights of the ovaries were decreased in females of the mid-dose group.

-       The absolute weight of the testis was decreased in mid-dose males. 

The main gross finding in animals of the mid-dose group (and in intercurrently killed animals of the high-dose group) were enlarged intestines with green/watery contents, a pale and/or green appearance of the liver and kidneys, small epididymides and seminal vesicles, enlarged dark spleen, small thymus and a variety of changes in the stomach.

 

Microscopic examination revealed histopathological changes in the kidneys, the liver and the spleen.

-       The histopathological changes in the kidneys were characterised by tubular necrosis and degeneration, tubular epithelial cell karyomegaly and accumulation of brown pigment. These changes were mainly present in the mid-dose animals. However, tubular epithelial brown pigment was also noted in the kidneys of 6/10 low-dose males.

-       The changes in the liver were accumulation of periportal macrophages, especially in the mid-dose males, hepatocellular karyomegaly, brown pigment accumulation, bile duct hyperplasia and (multi)focal infiltration of mononuclear inflammatory cells. These changes were mainly present in the mid-dose animals. However, mononuclear cell infiltrate was also noted in the liver of 6/10 low-dose males and 3/10 low-dose females.

-       The changes in the spleen were accumulation of brown pigment and accumulation of macrophages in the white pulp in animals of the mid-dose group.

The decedent high-dose animals were subjected to microscopical examination only on the basis of macroscopic observations. The microscopic observations in this group confirmed the above histopathological changes.

Based on the histopathological effects in liver and kidneys noted in animals of the low-dose group, the no-observed-effect level (NOEL) for parental toxicity was lower than 150 mg/kg bw/day. However, because only limited effects were observed in the low-dose group, it was speculated that the NOEL was close to 150 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well performed and reported GLP study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

When comparing the results of this study with EDTA-CuNa2 with repeated dose studies of other metal chelates (see read across document in section 13), it was concluded that EDTA-CuNa2 was slightly more toxic (LOAEL of 150 mg/kg bw) than the other metal chelates (NOAELs >= 500 mg/kg bw). In addition, it was also concluded, based on the limited effects observed at 150 mg/kg bw, the NOAEL would be close to 150 mg/kg bw.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Well performed and reported GLP study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Because in this subchronic study effects were limited at a dose level of 150 mg/kg bw, no classification is needed for STOT repeated exposure.