pentapotassium 2,2’,2’’,2’’’,2’’’’-(ethane-1,2-diylnitrilo)pentaacetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentasodium (carboxylatomethyl)iminobis(ethylenenitrilo)tetraacetate

EC Number:

205-391-3

EC Name:

Pentasodium (carboxylatomethyl)iminobis(ethylenenitrilo)tetraacetate

Cas Number:

140-01-2

IUPAC Name:

pentasodium 2,2',2'',2''',2''''-(ethane-1,2-diylnitrilo)pentaacetate

Details on test material:

Trilon C Flussig (containing DTPA pentasodium salt in water)
Degree of purity: > 43.7% pentasodium DTPA

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Sexually mature, virgin Wistar rats (Chbb :THOM (SPF)) supplied by Karl THOMAE, Biberach an der Riss, FRG, which were free from clinical signs of disease, were used for the investigations. This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be sensitive to substances with a teratogenic potential.
After randomization the rats were identified uniquely by ear tattoo. The unit digit of the animal number was tattooed on the outside of a rat's left ear and
the ten digit on the inside of the left ear and the hundred digit was tattooed into the right ear.
During the study period, the rats were housed singly in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 800 cm2). The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were accommodated in fully airconditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%. There were no deviations from these limits .
The day/night rhythm was 12 hours (12 hours light from 6.00 to 18.00 hours and 12 hours darkness from 18.00 to 6.00 hours).
Before the study started, the room was completely disinfected using a disinfection apparatus ("AUTEX" fully automatic final disinfecting apparatus using formaldehyde and ammonia). In general, each week the walls and the floor were cleaned with water containing about 0.5g Mikro-Quat (supplied by SCHÜLKE & MAYR GmbH, FRG).
The food used was qround Kliba 343 feed rat/mouse/hamster supplied by KLINGENTALMÜHLE AG, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy), as was drinking water of tap water quality from water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance solutions were prepared in intervals of not more than 4 days during the administration period, because the stability of the test substance solutions over a period of 4 days had been verified analytically . For the preparation of the solutions, an appropriate amount of the test substance was weighed in a volumetric flask, subsequently topped up with doubly distilled water and intensively shaken.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance over the study period was proven by reanalysis.
The results of the analyses of the aqueous solutions of test substance confirmed the correctness of the prepared concentrations.

Details on mating procedure:

The female rats were 61 - 70 days old when supplied. After an acclimatization period of at least 5 days, 1 - 4 untreated female rats were mated with one untreated fertile male animal of the same breed. Mating took place from about 16 .00 hours to about 7.30 hours on the following day . If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized. This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.). At the beginning of the study (day 0, detection of sperm), the rats were 83 - 101 days old. Their mean weight was approx. 256 g. On day 0, the animals were assigned to the different test groups according to a randomization plan.
The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 6 to day 15 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 6 p.c.).
On day 20 p.c., all females were sacrificed in a randomized order and examined macroscopically. The fetuses were removed from the uterus and further investigated with different methods.

Duration of treatment / exposure:

day 6 through day 15 post coitum

Frequency of treatment:

daily

Duration of test:

until day 20 post coitum

No. of animals per sex per dose:

22 pregnant female rats/group

Control animals:

yes, concurrent vehicle

Details on study design:

In a range-finding study, Trilon C liquid was administered to pregnant Wistar rats by gavage daily for a period of 10 days (days 6 - 15 post coitum) in doses of 200, 400 and 800 mg/kg body weight/day. For comparison, a group of sham-treated (doubly distilled water) animals was used as a control.
The primary aim of this range-finding study was to find a dose which should induce "some overt maternal toxicity such as slight weight loss, but not more than 10 per cent maternal deaths" and could be used as the highest dosage for the following full-scale prenatal toxicity study. Therefore, the dams were already sacrificed on the day following the last substance administration (i.e. day 16 p.c.). Shortly before sacrifice blood and urine samples were taken for hematological and/or clinicochemical examinations. At necropsy liver, kidneys and uterus (unopened) were weighed and the dams assessed by gross pathology. The fetuses were dissected from the uterus and subjected to a rough external examination.
The following findings were obtained and assessed as being possibly substance-related:
80/800 mg/k body weight/day: - statistically significantly lower alkaline phosphatase values
40/400 mg/kq body weight/day: - statistically significantly lower alkaline phosphatase values
20/200 mg/kg body weight/day: - no substance-related effects on dams, gestational parameters or fetuses
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In the second study, Trilon C liquid was administered to 5 male and 5 female Wistar rats in the drinking water in doses of 600 ppm (test group 1), 3,000 ppm (test group 2) and 12,000 ppm (test group 3) for 4 weeks. For comparison, a group of untreated animals (5 males, 5 females) was used as a control (test group 0).
Food consumption, water consumption and body weight were determined weekly. The state of health was checked each day. During the weekly weighing the animals were subjected to an additional comprehensive clinical examination. Urinalyses as well as clinicochemical and hematological examinations were carried out during the final week of dosing. All animals were assessed by gross pathology, followed by a histopathological examination.
The administration with Trilon C liquid to Wistar rats in the drinking water for 4 weeks caused the following findings:
12,000 ppm (about 1,775 mg/kg body weight): - Discoloration of the feces in both sexes - Decreased food consumption in both sexes - Significantly decreased body weight and body weight change in males during the course of the study, in females in the first two weeks, only - Increase in alanine aminotransferase in the males - Increase in specific gravity, renal epithelial cells and casts in the urines of individual males - Dark yellow colored urines in individual males - Decrease in urine volume in both sexes - Decrease in alkaline phosphatase in the females - Significantly decreased mean terminal body weight of the male test animals. The female test animals showed a tendency for a decreased meañ terminal body weight. - Transitional cell hyperplasia in the urinary bladder of 4 male and 2 female test animals.
3,000 ppm (about 420 mg/kg body weight): - Significantly decreased body weight change in the males in the last test week - Increase in alanine aminotransferase in the males - Decrease in alkaline phosphatase in the females.
600 ppm (about 75 mg/kg body weight): - No substance-induced changes
-----
Taking into consideration the results of the above mentioned studies, the following doses were fixed for the full-scale prenatal toxicity study in rats with Trilon C liquid: 100 mg/kg body weight - as the expected no observed effect level; 400 mg/kg body weight - as the intermediate dose level; 1000 mg/kg body weight - as the dose with maternally toxic effects at which findings in fetuses may also be obtained.

Examinations

Maternal examinations:

Food consumption, body weight data, corrected body weight gain (net maternal body weight change), clinical symptoms, mortality.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: yes
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorptions: yes
- Number of late resorptions: yes

Fetal examinations:

Examination of the fetuses after dissection from the uterus, soft tissue examination of the fetuses, skeletal examination of the fetuses, evaluation criteria for assessing the fetuses

Statistics:

Examinations of dams and fetuses

Historical control data:

yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
details see below

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
details see below

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Test group 3 (1,000 mg/kg body weight/day): statistically significantly reduced food consumption during the first half of the treatment period; if calculated for the whole treatment period (days 6- 15 p .c .) about 7% less food intake than the controls / statistically significantly lower body weights than the controls on days 17 and 20 p.c / statistically significant impairment in body weight gain; if calculated for the total treatment period about 21% less weight gain than the controls / about 12% lower corrected body weight gain than the controls / dark-yellow discoloration of the feces in all females during most days of the treatment period and the first days of the posttreatment phase / statistically significantly lower mean gravid uterus weight (about 21% lower than in the control group) / statistically significant reduction of the mean number of live fetuses/litter (11.9 vs. 14.3 in the control group); slightly increased number of resorptions and insignificantly increased postimplantation loss value / about 8% lower mean fetal body weights in comparison to the control group / statistically significantly increased malformation rate (15.4% affected fetuses/litter vs. 3.5% affected fetuses/litter in the control group), predominantly caused by an increased occurrence of skeletal malformations / statistically significantly increased rate of fetuses with skeletal variations (78.4% affected fetuses/litter vs. 49.6% affected fetuses/litter in the control group) and retardations (78.0% affected fetuses/litter vs. 47.4% affected fetuses/litter in the control group).

Test qroup 2 (400 mg/kg body weight/day): statistically significantly increased rate of fetuses with skeletal retardations (63.8% affected fetuses/litter vs. 47.4% affected fetuses/litter in the control group).

Test group 1 (100 mg/kg body weight/day): no substance-related effects on dams, gestational parameters or fetuses.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this full-scale study, Trilon C liquid caused clear signs of maternal toxicity at 1,000 mg/kg body weight/day, while 100 or 400 mg/kg body weight/day were tolerated by the dams without any substance-induced findings. Most, but not all signs of maternal toxicity which occurred at 1,000 mg/kg body weight/day have to be associated with the lower number of live fetuses/dam and the lower mean fetal body weights in this group. At the highest dose level, clear signs of developmental toxicity, including teratogenicity, were obtained. The fetal weights were statistically significantly lower than in the concurrent control. The malformation rate was clearly increased, which was primarily caused by a higher rate of 1,000 mg/kg fetuses with malformations of the vertebral column and the sternebrae. Furthermore, statistically significantly more fetuses showed skeletal variations and retardations. At the intermediate dose level (400 mg/kg body weight/day) an increased rate of fetuses/litter showed skeletal retardations, the only sign of developmental toxicity which was recorded at this dose level. At 100 mg/kg body weight/day, however, no signs of embryo-/fetotoxicity, especially no substance-induced indications of teratogenicity, were observed.