Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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 according to GLP
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
premating period was extended from 2 to 10 weeks to cover a full sperm cycle
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 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
Details on mating procedure:
At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occurred or 1 week had elapsed. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant and females that did not show evidence of copulation were killed not earlier than 21 days after the last day of the mating period.
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 (parental animals)
Details on study schedule:
No
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
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays andpublic holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded. Animals showing signs of severe debility or intoxication, particularly if death appeared imminent, were killed.
In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on 10 (if surviving) rats/sex/group rats (those with the lowest identification numbers) prior to the first exposure and then once weekly during the premating period. In week 9 (females) or 10 (males) of the premating period, the detailed clinical observations were included in the Functional Observation Battery.
Functional Observational Battery (FOB) tests and spontaneous Motor Activity Assessment (MAA) were performed in 10 (if surviving) rats/sex/group at the end of the premating period (females in week 9 and males in week 10).

BODY WEIGHT: Yes
The body weight of each animal was recorded at initiation of treatment (day 0). Subsequently, males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. At scheduled necropsy, the male and female animals were weighed in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION: Yes
Feed consumption was measured per cage by weighing the feeders. The consumption was measured from day 0 over successive weekly periods (coinciding with the body weight measurements). During lactation, feed consumption was determined from day 1 to 4.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):
Not measured
Sperm parameters (parental animals):
Parameters examined:
Epididymal sperm motility, count and morphology
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis in the control-, low- and mid-dose group (from ten males with the lowest identification numbers in the control and low-dose group, and from the nine surviving males in the mid-dose group). Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured. In addition, a smear of the sperm solution was prepared and stained for the same males of the control-, low- and mid-dose group, but only the smears of the (10) control- and (9) mid-dose males were examined microscopically for morphology. Because no treatment-related changes were observed, examination of sperm morphology was not conducted in the low-dose group.

Testicular sperm count
At scheduled necropsy, the left testis of males of the control-, low- and mid-dose group (ten males with the lowest identification numbers in the control and low-dose group, and nine surviving males in the mid-dose group) were placed on dry ice and subsequently stored in a freezer (<-70°) for later determination of the number of homogenization-resistant spermatids. The daily sperm production was calculated as ‘number of spermatozoa per gram testicular parenchyma. Because no treatment-related changes were observed, evaluation of homogenization-resistant spermatids was not conducted in the low-dose group.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, because this screening study was ended on day 4 post-partum

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
At scheduled necropsy, all surviving male and female parent (F0) animals were sacrificed whilst under CO2/O2 anaesthesia and then examined macroscopically for pathological changes. Male animals were sacrificed after 90-days of treatment. Female animals were sacrificed at day 4 of lactation. Necropsy was also performed on animals that died intercurrently or that were killed in moribund condition.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

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 sex organs listed below :
-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).
- 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).
- ovaries, uterus, testes, epididymides, seminal vesicles, prostate, coagulating glands
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic) externally for gross abnormalities

GROSS NECROPSY
Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. At necropsy of the dams at day 4 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques. Pups were stored in a freezer for possible skeletal analyses

ORGAN WEIGHTS: not done

HISTOPATHOLOGY: not done
Statistics:
- Body weight and feed consumption data: one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Incidences of histopathological changes of parent animals: Fisher’s exact probability test.
- Litter data: Fisher’s exact probability test or Kruskal-Wallis nonparametric analysis of variance (see Table 16).
- Sperm parameters: one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests (epididymal and testicular sperm count and numerical sperm motility parameters) or Kruskal Wallis non parametric ANOVA followed by Mann Whitney U tests (motility parameters expressed as percentage and sperm morphology).
See also section 7.5.1.
Reproductive indices:
pre-coital time = time between the start of mating and successful copulation
duration of gestation = time between gestation day 0 and day of delivery
mating index = (number of females mated/number of females placed with males) x 100
male fertility index = (number of males that became sire/number of males placed with females) x 100
female fertility index = (number of pregnant females/number of females placed with males) x 100
female fecundity index = (number of pregnant females/number of females mated) x 100
gestation index = (number of females with live pups / number of females pregnant) x 100
Offspring viability indices:
live birth index = (number of pups born alive/number of pups born) x 100
viability index day 1-4 = (number of pup surviving 4 days/number of liveborn on day 1) x100
pup mortality day 1 or 4 = (number of dead pups on day 1 or 4/total number of pups on day 1 or 4) x 100
sex ratio day 1 or 4 = (number of live male fetuses or pups on day 1 or 4/ number of live fetuses or pups on day 1 or 4) x 100
pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
number of lost implantations = number of implantations sites - number of pups born alive
post-implantation loss = [(number of implantation sites - number of pups born alive)/ number of implantation sites] x 100.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
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 (PARENTAL ANIMALS): no effects (gavage)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): not measured

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no effects

ORGAN WEIGHTS (PARENTAL ANIMALS):
- 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 (PARENTAL ANIMALS): 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 (PARENTAL ANIMALS): 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.





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: slight liver and kidney changes at the level of 150 mg/kg bw/day; based on these limited effects it is expected that the NOAEL is close to 150 mg/kg bw
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction effects at the level of 500 mg/kg bw. At the next higher level of 1500/1050 mg/kg bw mortality occurred.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): no effects

SEXUAL MATURATION (OFFSPRING): not done, pups were necropsied on day 4 post partum

ORGAN WEIGHTS (OFFSPRING): not done

GROSS PATHOLOGY (OFFSPRING): no abnormaities

HISTOPATHOLOGY (OFFSPRING): not done

OTHER FINDINGS (OFFSPRING): none
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effects at this level.
Reproductive 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. Because fertility parameters, reproductive performance and development were not affected by the test substance, the NOEL for reproduction and developmental effects was placed at ≥ 500 mg/kg bw/day.

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 findings are indicated in section 7.5.1.

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.

There were no effects of the test substance on male fertility parameters (epididymal sperm motility, sperm count and sperm morphology, and testicular sperm count and daily sperm production). No treatment-related effects were seen in any of the reproductive or developmental indices.

 

Based on the 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. Because fertility parameters,reproductive performance and development were not affected by the test substance, the NOEL for reproduction and developmental effects was placed at ≥ 500 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well performed and reported GLP study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

See also the read across document in section 13.

In a 2 year feeding study on Wistar rats including reproductive and lactation experiments in four successive generations groups of 25 male and 25 female animals were exposed to EDTA-CaNa2 at dietary levels providing daily doses of approximately 50, 125, and 250 mg/kg bw (Oser et al., 1963). No significant differences in behavior or appearance nor adverse effects on the growth or on the longevity of the rats in any of the generations or among the various dose levels were reported. Evaluations of various tissues and organs (weight, histopathologic examinations) including gonads (testes) gave negative results even in the high dose group. Criteria for reproductive and lactational effects were evaluated as proportion of matings resulting in pregnancy (fertility index), proportion of pregnancies resulting in live litters (gestation index), proportion of pups that survive 4 days or longer (viability index), and proportion of rats alive at 4 days that survive to weaning. Poor responses with respect to some of the criteria of reproductive performance occurred occasionally but were not correlated with dosage or with the number of generations through which dosage continued. The overall data for two matings in the four successive generations did not give evidence for significant treatment related differences in either of these indexes. The authors concluded that the No Observed Adverse Effect Level of EDTA-CaNa2 was observed as measured by any of the usual indices of reproduction or lactation efficiency even under the stresses of repeated pregnancies and lactation. The NOAEL derived from this study is therefore at least 250 mg/kg bw/day for the parent and F1 to F3 generation.

Studies with EDTA-Na2H2 were not taken into consideration for setting a NOAEL because of methodological flaws. Studies with other metal-chelates (EDTA-MnNa2 and DTPA-FeNaH) showed effects on fertility only at very levels of 1500 mg/kg bw but not at 500 mg/kg bw. But although effcets on sperm were

seen at the highest dose of EDTA-MnNa2 or DTPA-FeNaH tested (1500 mg/kg bw), it did not result in effects on reproduction as there were no changes in reproductive performance in animals of these groups.

Short description of key information:

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. Because fertility parameters and reproductive performance were not affected by the test substance, the NOEL for reproduction was placed at  500 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:

Well performed and reported GLP study

Effects on developmental toxicity

Description of key information

Based on the effects in liver in females of the low-dose group, the no-observed-effect level (NOEL) for maternal toxicity was lower than 150 mg/kg bw/day. Because development was not affected by the test substance, the NOEL for developmental effects was placed at  500 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
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 according to GLP
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 422
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
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 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.
Details on mating procedure:
At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occurred or 1 week had elapsed. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant and females that did not show evidence of copulation were killed not earlier than 21 days after the last day of the mating period.
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
Duration of test:
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.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
This dose was lowered to 1050 mg/kg bw/day due to severe toxicity
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
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS / HAEMATOLOGY / CLINICAL CHEMISTRY: Yes
See section 7.5.1.

BODY WEIGHT: Yes
The body weight of each animal was recorded at initiation of treatment (day 0). Subsequently, males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. At scheduled necropsy, the male and female animals were weighed in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION: Yes
Feed consumption was measured per cage by weighing the feeders. The consumption was measured from day 0 over successive weekly periods (coinciding with the body weight measurements). During lactation, feed consumption was determined from day 1 to 4.

WATER CONSUMPTION: No

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No as females were allowed to litter
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
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).
- Incidences of histopathological changes of parent animals: Fisher’s exact probability test.
- Litter data: Fisher’s exact probability test or Kruskal-Wallis nonparametric analysis of variance (see Table 16).
See for other statistical tests, section 7.5.1.
Indices:
- gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m= (number of pup surviving m days/number of liveborn on day n) x100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100
Historical control data:
Not included.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In females exposed to 1500/1050 mg/kg: mortality:
In females exposed to 500 mg/kg: effects on haematology, clinical chemistry, organ weight changes, and histopatologocal changes in liver, kidneys and spleen.
In females exposed to 150 mg/kg: slight histopathological changes in liver
Dose descriptor:
NOAEL
Effect level:
ca. 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no developmental effects at 500 mg/kg bw/day. At the next higher level of 1500/1050 mg/kg bw maternal mortality occurred.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the effects in liver in females of the low-dose group, the no-observed-effect level (NOEL) for maternal toxicity was ca. 150 mg/kg bw/day. Because development was not affected by the test substance, the NOEL for developmental effects was placed at ≥ 500 mg/kg bw/day.
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 findings are indicated in section 7.5.1.

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.

There were no effects of the test substance on male fertility parameters (epididymal sperm motility, sperm count and sperm morphology, and testicular sperm count and daily sperm production). No treatment-related effects were seen in any of the reproductive or developmental indices.

 

Based on the effects in the liver in females of the low-dose group, the no-observed-effect level (NOEL) for maternal toxicity was lower than 150 mg/kg bw/day. Because development was not affected by the test substance, the NOEL for developmental effects was placed at ≥ 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Study duration:
chronic
Species:
rat
Quality of whole database:
Well performed and reported GLP study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

See also read across document in section 13.

EDTA and four of its salts were evaluated for their teratogenic potential in CD albino rats (Schardein et al., 1981). Groups of 20 females were treated by gavage during g.d. 7 to 14 with 1,000 mg EDTA/kg bw/day as well as with equimolar doses of disodium, trisodium, calcium disodium and tetrasodium edetate. The dose level had been selected from preliminary studies with edetic acid in which there had been some evidence of both maternal and fetotoxicity under the same experimental conditions. For the dams significant drug-related reactions including diarrhea and depression of activity were reported. The former occurred in all drug groups with highest incidences for tetrasodium edetate (90%) and edetic acid (80%) and lowest incidence for calcium disodium edetate (10%). Three dams died during treatment with disodium edetate. Besides slightly decreased food intake in all test groups, treatment with all of the test compounds caused reduced weight gain in the dams during the treatment period. The mortality index of offspring in all treated groups as measured by postimplantation loss was comparable to that of the vehicle and untreated control group. None of the test compounds significantly affected litter size at term or mean fetal body weight when compared to either control. Fetuses were examined for external, visceral and skeletal anomalies. Incidental findings of skeletal anomalies did not reveal a definitive pattern regarding treatment with a particular compound. The authors stated that under these experimental conditions no teratogenic effects were evidenced even at maternally toxic doses.

In a further developmental study pregnant Sprague-Dawley rats were exposed during various periods of gestation to purified diets adjusted to either 100 or 1,000 ppm zinc (provided as zinc carbonate) and containing 2 or 3% Na2EDTA corresponding to 1000 or 1500 mg/kg bw daily intake (Swenerton and Hurley, 1971). The groups of 8 to 16 females had been set on the control diet at least 5 days before breeding and mated to normal stock-fed males. The evaluation of treatment related effects to the dams was not indicated in this study, except for the report on moderate to severe diarrhea in all females that were fed diets containing Na2EDTA. While obviously complete reproductive failure occurred with the 3% Na2EDTA/100 ppm zinc diet fed during g.d. 0-21, with the 2% Na2EDTA/100 ppm zinc diet reproductive outcome was essentially comparable to that of controls, however with lower mean body weight of the pups and with 7% malformed of the fullterm fetuses. Exposure to the 3% Na2EDTA/100 ppm zinc diet during the period of g.d. 6-14, and 6-21 resulted in respectively 40% and 54% dead or absorbed fetuses, reduced number of dams with live pubs, clearly reduced mean fetal body weight and ratios of respectively 87% and 100% malformed living offspring. Gross malformations comprised cleft palate, severe brain deformities, eye defects, micro- or agnathia, syndactyly, clubbed legs and tail anomalies. The reported fetotoxic and teratogenic effects were similar to those from earlier experiments with zinc deficient diets administered to pregnant rats for various periods of during gestation (Hurley, 1966). In contrast, the live offspring of dams fed 3% Na2EDTA supplemented with 1,000 ppm zinc from g.d. 6-21 did not exhibit any malformations, and the mean number of live pups/litter and the mean fetal body weight were comparable to those of controls. The authors concluded from this study that Na2EDTA ingested during pregnancy was teratogenic, whereas supplementation with zinc prevented the detrimental effects of EDTA. It was suggested that the congenital anomalies caused by EDTA were due specifically to zinc deficiency. This was also supported by zinc analyses of fetuses (Hurley and Swenerton, 1966), where clearly lower zinc contents were found in fetuses from deficient mothers in comparison to those from zinc supplemented dams, indicating that the reported effects rather occur because of a direct lack of zinc in fetal tissues than from indirect effects of maternal metabolism on fetal development.

The toxic and teratogenic effects of Na2EDTA were studied in female CD rats following different routes of administration (dietary, gavage, s.c) during g.d. 7-14 (Kimmel, 1977). Dietary exposure to 3% Na2EDTA amounting to an average dose of 954 mg Na2EDTA/kg bw/day resulted in reduced food intake, severe diarrhea and severe weight loss in the dams during treatment and produced a significant proportion of fetal deaths (about 33% resorptions/litter), significantly lower average fetal weight and gross external, internal and skeletal malformations in about 71% of the survivors. Treatment with 1,500 or 1,250 mg Na2EDTA/ kg bw/day administered by gavage (respectively 625 mg/kg and 750 mg/kg twice daily) resulted in severe toxicity to the dams (7 out of 8 animals died in the 1,500 mg dose group), in particular 36% maternal deaths, significantly reduced weight gain, and diarrhea in the 1,250 mg dose group and a significantly higher proportion of (about 21%) malformed survivors. Treatment with 375 mg/kg bw administered subcutaneously produced signs of severe pain (vocalisations and shock) to the dams and resulted in 24% maternal deaths, significantly reduced food intake and maternal weight loss during the period of treatment. Fetal toxicity (about 32% resorptions/litter, significantly reduced fetal weight) and a rate of about 4% malformed survivors/litter were reported for this route of application.

EDTA-MnNa2 showed developmental effects only at 1500 mg/kg bw, whereas no developmental effects were observed at 1500 mg/kg bw DTPA-FeNaH. The effects observed following treatment with EDTA-MnNa2 consisted of a decreased number of females with live born pups, decreased number of (live) pups, increased postimplantation loss. No such effects were seen at 500 mg/kg bw. Because of the higher affinity of EDTA for Zn it can also be expected that at the high level of 1500 mg/kg bw, sufficient Mn will be exchanged for Zn and as such Zn-deficiency may also occur as has occurred with the 'empty' (non-metal containing) chelates.

Justification for selection of Effect on developmental toxicity: via oral route:

Well performed and reported GLP study

Justification for classification or non-classification

Effects on reproduction and effects on fetal development have not been observed at levels up to 500 mg/kg bw. At the next higher level of 1500/1050 mg/kg bw significant mortality occurred before mating took place. Therefore, based on the results obtained in the studies and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, classification with regard to toxicity to reproduction is not required.