Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: dilution in water
Details on test material:
Chemical name: Copper disodium ethylenediamine tetraacetate
Purity: 92.7%
Batch no: CFC 10334
Expiry date: 31 August 2012

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects.

Effect levels (fetuses)

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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.