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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The administration route is intraperitoneal and not oral as in the guidelines for developmental toxicity study but the study is in general comparable to the guidelines and well documented

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Despite the effectiveness of CDTA in the treatment of zinc, lead and manganese intoxications, at the date of the study, data on the toxic effects of this chelator -including the developmental toxicity- have not been available, therefore the study was performed to evaluate the embryotoxic and teratogenic potential of CDTA administered to mice during organogenesis. The study is comparable to guideline for developmental toxicity study and is well documented, but intraperitoneal route was used as administration route instead of the oral route as in the guideline.
GLP compliance:
not specified

Test material

1
Chemical structure
Reference substance name:
trans-cyclohexane-1,2-dinitrilotetraacetic acid
EC Number:
236-308-9
EC Name:
trans-cyclohexane-1,2-dinitrilotetraacetic acid
Cas Number:
13291-61-7
Molecular formula:
C14H22N2O8
IUPAC Name:
2,2',2'',2'''-(cyclohexane-1,2-diyldinitrilo)tetraacetic acid
Test material form:
solid: particulate/powder
Details on test material:
Purity: > 99 %
Specific details on test material used for the study:
CDTA was purchased from Sigma Chemical Co. and administered as the disodium calcium salt, and solutions prepared by adding 2:1:1 molar ratio amounts of NaOH, CDTA and Ca(OH)2 to 0.9% saline.

Test animals

Species:
mouse
Strain:
Swiss
Details on test animals or test system and environmental conditions:
Mice (30-33 g) were quarantined for 1 week. Two female mice were mated with one male overnight and examined the following morning for presence of vaginal plug (gestation day 0). Food and tap water were available ad libitum throughout the study. Temperature and humidity in the animal rooms were kept at 22*+/- 2°C and 50+/-10% respectively. An alternating 12-hr cycle of light and darkness was followed.

Administration / exposure

Route of administration:
intraperitoneal
Details on exposure:
Solution concentrations were adjusted so that a 30 g mouse would receive a volume of 0.15 ml
Duration of treatment / exposure:
Injection once daily on days 6 to 15 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
270 other: mg/Kg
Dose / conc.:
540 other: mg/Kg
Dose / conc.:
1 080 other: mg/Kg
Remarks:
A fourth group of mice received i.p. injections of 0.9% NaC1 solutions (control group).
Details on study design:
Body weight, food consumption, and general appearance were monitored daily. On day 18 of gestation dams were weighed with gravid uterus and killed by an ether overdose. Liver, kidneys and placenta were removed, weighed, frozen and stored at -20° C until trace element analyses. All live fetuses were dissected from the uterus and evaluated for body weight, sex, and external abnormalities. Three fetuses from each dam were used to determine whole-fetuses analyses of calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and iron (Fe). The remaining fetuses in the litters were preserved intact in Bouin's solution and processed for the evaluation of visceral anomalies (Wilson, 1965), or fixed in 95% ethanol, macerated in 1% KOH, stained with Alizarin ruf S, and examined for skeletal malformations and variations (Staples and Schnell, 1964). For the determination of mineral concentrations, tissues were mixed with 65% HNO3 and heated under pressure at 190° C. The samples were then diluted with deionized water, filtered, and measured in a computer-controlled sequential inductively coupled plasma spectrometer (Zn, Cu, Fe), or in an atomic absorption spectrophotometer (Ca, Mg).

Examinations

Statistics:
CDTA-treated groups were compared with the control group at a level of significance of p < 0.05. Results of the quantitative continous variables were intercompared for the three treated groups and the control group by a one-way analysis of variance with significant F values further analyzed using the Student's t-test or the Mann-Whitney U-test. Incidence data were analyzed by the X2-test for independence, and the Fisher exact test for pairwise comparison.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Mortality:
mortality observed, treatment-related
Description (incidence):
Although there were no maternal deaths, no abortions, and no early deliveries at 270 and 540 mg CDTA/kg/day, severe maternal toxicity was observed in the 1080 mg/kg/day group. Fifty per cent (50.0%) of the assigned females died during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although the mean body weight gain during the pretreatment interval (days 0-6) was comparable between treated and control animals, maternal weight gain during the treatment (days 6-15) and posttreatment (days 15-18) periods was significantly decreased in the 1080 mg/kg/day group compared with the controls.
At sacrifice on day 18 of gestation, there were no significant differences in absolute body weight, gravid uterine weight, corrected body weight (body weight at termination minus gravid uterine weight), corrected body weight change (corrected body weight minus body weight on gestation day 0), or in absolute and relative liver and kidney weights in the dams exposed to 270 and 540 mg CDTA/kg/day.
A significant reduction (p < 0.001) in body weight at termination and in gravid uterine weight was observed at 1080 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly reduced in the 1080 mg/kg/day group during the dosing and post-dosing periods. In contrast, maternal weight gain and maternal food consumption were not affected by CDTA exposure at 270 and 540 mg/kg/day in the pretreatment, treatment and post-treatment intervals.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Treatment with CDTA during organogenesis significantly reduced the hepatic concentrations of Mg and Zn at 1080 mg/kg/day, whereas hepatic levels of Cu were increased at this dose. However, there were no differences in maternal liver, kidney and placenta Ca levels between treated and control mice. Intraperitoneal administration of CDTA significantly diminished maternal Fe levels at 270 (kidney), 540 (liver and kidney), and 1080 mg/kg/day (liver, kidney, and placenta).

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
No abortions at 270 and 540 mg CDTA/kg/day.
High percentage of abortions (18.7%) in the 1080 mg/kg/day group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The percentage post-implantation loss per litter was significantly increased at 1080 mg/kg/day versus control
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
No early deliveries at 270 and 540 mg CDTA/kg/day. High percentage of resorbed litters (37.4%) in the 1080 mg/kg/day group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
The number of late resorptions was significantly increased at 1080 mg/kg/day versus control
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The number of dead fetuses was significantly increased at 1080 mg/kg/day versus control

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
dead fetuses
early or late resorptions
food consumption and compound intake
maternal abnormalities
mortality
number of abortions
pre and post implantation loss
total litter losses by resorption

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Average fetal body weight per litter decreased significantly in the 1080 mg/kg/day group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of live fetuses per litter was significantly depressed at 1080 mg/kg/day when compared with controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no significant differentes among dose groups in the number of implantation sites per litter or in the sex ratio.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Incomplete ossification of sternebrae, parietal, frontal and occipital bones were the most frequent developmental variations observed at 1080 mg/kg/day.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Exencephaly, abnormally small kidneys, and enlarged heart auricles were the predominant malformations observed at 1080 mg/kg/day.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Although a non-statistically significant trend of lower Ca, and Fe fetal mineral concentrations was observed in CDTA-exposed groups when compared with controls, fetal mineral levels were only affected significantly at 270 mg/kg/day (Fe) and 1080 mg/kg/day (Cu).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
visceral/soft tissue: cardiovascular
visceral/soft tissue: central nervous system
other: Kidneys

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 080 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified

Applicant's summary and conclusion

Conclusions:
The results of this study show that treatment with CDTA produced severe maternal toxicity (lethality) in mice at 1080 mg/kg/day. Other maternal signs of toxicity were also observed at this dose, and consisted of decreased body weight gain and food consumption seen on days 6-15, 15-18, and 0-18 of gestation was always accompanied by a significant reduction in body weight gain.
Embryo/fetal toxicity, including teratogenicity, was also observed at 1080 mg CDTA/kg/day, expressed as a significant increase in the number of resorptions and fetal deaths, a decrease in the number of live fetuses per litter, as well as reduced fetal body weight per litter. At this dose, an increase in the number of litters affected with fetal abnormalities was also observed.
In contrast, embryo/fetal toxicity of CDTA was not seen at 270 and 540 mg/kg/day when the unit of comparison was the pregnant female or the litter. However, there was a significant increase in the number of fetuses with exencephaly at 540 mg/kg/day (on the basis of affected fetuses).
With regard to the effects of CDTA on mineral metabolism, although this chelator is known to increase the excretion and to decrease the tissue concentrations of a number of heavy metals (Tandon and Singh, 1975; Domingo et al., 1989a,b; Llobet et al., 1989), in the present study CDTA did not induce remarkable changes in the levels of minerals in maternal liver, kidney, or placenta. Moreover, although CDTA caused a slight effect on the fetal concentrations of Cu and Fe, these changes were sporadic and not dose-related. Therefore, the effects of CDTA on mineral metabolism would not explain the induction of the teratogenic effects observed at 1080 mg/kg/day.
Executive summary:

The current study revealed maternal and developmental toxicity in Swiss mice following i.p. administration of CDTA at doses as high as 1080 mg/kg/day during organogenesis. The no observable adverse effect level (NOAEL) for maternal and embryo/fetal toxicity was 540 mg/kg/day, although the NOAEL for teratogenicity would be 270 mg/kg/day when taking the fetus as the unit of comparison. Although the mechanism of CDTA-induced embryo/fetal toxicity is unknown, the chelator produced developmental toxicity in mice only in the presence of maternal toxicity. The decreases in maternal and fetal concentrations of some elements observed following CDTA exposure would probably be the consequence of a direct chelation of the affected elements by the drug, and they should not be the major reason for CDTA-induced developmental toxicity.