Etidronic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1971

Report date:

1970

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Dosing from day 2-16 instead of day 6-18; uterine weights (does) and sex ratios (offspring) were not evaluated

GLP compliance:

no

Remarks:

Conducted prior to adoption of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium dihydrogen (1-hydroxyethylidene)bisphosphonate

EC Number:

231-025-7

EC Name:

Disodium dihydrogen (1-hydroxyethylidene)bisphosphonate

Cas Number:

7414-83-7

Molecular formula:

C2H8O7P2.2Na

IUPAC Name:

disodium dihydrogen (1-hydroxyethane-1,1-diyl)bis(phosphonate)

Test material form:

not specified

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Virgin New Zealand does, 5-6 months of age and about 4 kg bodyweightat the time of insemination, were randomly distributed into groups of 25 (pre-study) or 20 (main study) does on the basis of weight and litter after an 18-day holding period. Water and food (Purina Rabbit Chow) was available ad libitum. They were housed in standard stainless steel rabbit cages, one female to a cage.

ENVIRONMENTAL CONDITIONS:
Room temperature was maintained at 23 +/- 1°C, and relative humidity at 50 +/- 5%. Lighting was on a 12 hour cycle and background music was employed to equalize ambient noises.

Administration / exposure

Route of administration:

other: oral/gavage in the pre-study, oral/feed and oral/gavage in the main study

Vehicle:

other: drinking water (gavage application, pre-study and main study), no vehicle (incorporation in food, main study)

Details on exposure:

PRE-STUDY:
Except for the untreated control group, the females were dosed with either the test material in water or water alone on days 2 through 16 (day inseminated = day 1). Each doe recieved 2 mL of fluid per kg bw. Dosing was commenced prior to implantation (day 7) so that any possible preimplantation effects might be revealed. The compound was given as an aqueous mixture by intubation.

MAIN STUDY:
In the main study, doses of 25, 50 or 100 mg/kg bw/d were used. The compound was incorporated into ground rabbit feed which was then repelleted and fed to the rabbits from day 2 through day 16 of pregnancy.
To determine whether the ingestion of etidronate in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/d of the material by stomach tube just as was done in the Pre-study. Control groups, untreated and water-treated, were included.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Females were artificially inseminated by the method of Gibson et al (1966).

Duration of treatment / exposure:

GD 2-16

Frequency of treatment:

daily

Duration of test:

29 d

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

Pre-study: 25
Main study: 20

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

PRE-STUDY:
The females were artificially inseminated by the method of Gibson et al (1966) and except for the untreated controls, were dosed with either the test material in water or water alone on days 2 through 16 (day inseminated = day 1). Each doe recieved 2 mL of fluid per kg bw. Dosing was commenced prior to implantation (day 7) so that any possible preimplantation effects might be revealed. The compound was given as an aqueous mixture by intubation. Because of the toxicity to the dam. however, the 500 mg/kg bw/d dosage was later reduced to 250 mg/kg bw/d.
The pregnant does were sacrificed on day 29 and examined for resorptions, corpora lutea and implantations. The fetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the fetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the fetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

MAIN STUDY:
Due to the maternal toxicity from the 500 mg/kg bw/d dose of disodium etidronate in the pre-study, doses of 25, 50 or 100 mg/kg bw/d were used. The compound was incorporated into ground rabbit feed (Purina Rabbit Chow) which was then repelleted and fed to the rabbits from day 2 through day 16 of pregnancy.
To determine whether the ingestion of etidronate in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/d of the material by stomach tube just as was done in the pre-study. Control groups, untreated and water-treated, were included.
To reduce the possibilty of a dietary bias, all rabbits fere fed ground feed which had been repelleted during both the orientation period and the 29 days of gestation.The feed consumed by 25 does was measured for 14 days during the orientation period to establish the dietary level of test material to be incorporated into the diet, and later the feed consumed from day 2 through day 16 of pregnancy was measured for all does, both experimental and control. The latter was done not only to determine the actual amount of disodium etidronate ingested, but also to determine whether the stress of intubing the rabbits affected feed intake.
All does were inseminated by the method of Gibson et al., 1966 and weighed on that day and again on day 29 of pregnancy. The former weights were used to calculate the dose levels, whether given by intubation or incorporated into the feed. Those given the disodium etidronate by stomach tube and water-treated controls were weighed daily, since this caused no additional trauma, in order to monitor the weight gains throughout gestation.
Likewise, the laparotomies and collection of data were done as described in the pre-study.

Examinations

Maternal examinations:

Pregnant does were examined for resorptions, corpora lutea and implantations. The body weight was recorded on GD0 and GD29. The food intake was recorded daily.

Fetal examinations:

The fetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the fetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the fetuses were examined for soft-tissue anomalies, either by histological methods or by freehand sectioning (Wilson, 1965).

Statistics:

Analyses of variance were done on the appropriate data (Snedecor, 1946), and the partitioning was done by the Tukey "minimum difference" test as described by Scheffe (1952).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Does receiving 500 mg/kg bw/d died after day 4 or 5 of dosing (pre-study). Consequently, the 500 mg/kg dosage was later reduced to 250 mg/kg/day.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day active acid

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day active acid

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

PRE-STUDY:

 

New Zealand does were successfully inseminated in 81% of the attempts, although this varied significantly from 100% in the water-dosed control to 68% in the group treated with 100 mg/kg bw/d of disodium etidronate.

Does recieving 500 mg/kg bw/d died after day 4 or 5 of dosing. However, 4 females that recieved only 3 doses before having the dose reduced to 250 mg/kg bw/d survived to term. They were not included in the statistical analyses, but their data were included, so that limited comparisons between the 2 doses could be made.

There were no significant differences in the number of corpora lutea, implantations, resorptions or live fetuses or their weights. The number of live fetuses, however, was reduced in the group given 100 mg/kg bw/d.

Of 304 control fetuses examined, 7 exhibited frank abnormalities (table 1). These included heart, eye and urogenital defects. 6 of these were in the water-dosed control group. 2 Fetuses out of 99 from mothers intubed with 100 mg/kg bw/d were defective, while none were seen in the 18 fetuses from mothers intubed with higher doses.

30 to 40% of all fetuses had supernumerary ribs; most of these were bilateral. Variations in the sternebrae were quite common, with atrophy of the fifth sternebrae occurring with the greatest frequency.

Histopathologic examination of the sacrificed dams showed renal tubular degeneration in all those given 500 and 250 mg/kg bw/d and in 20% of those dosed with 100 mg/kg bw/d. Since 50% of the control animals were similarly affected, this condition was not attributed to the etidronate treatment.

 

TABLE 1 (PRE-STUDY): EFFECTS OF INTUBATED DISODIUM ETIDRONATE ON REPRODUCTION AND TERATOGENY OF NEW ZEALAND RABBITS

 

	Untreated control	H2O control	100 mg/kg	500 mg/kg****
No. pregnant	19	22	15	-
Conception rate	82.6	100.0	68.2***	-
No. females dead	1	1	2	20
Mean corpora lutea	9.7	9.6	10.3	11.3
Mean resorptions	1.2	1.1	1.4	3.3
Mean live fetuses	8.0	7.8	6.7	9.0
Mean fetus weights (g)	26.4	25.8	26.3	-
No. fetuses examined	146	158	99	18
No. with soft-tissue defects	1	6	2	0
No. with skeletal defects	0	0	0	0
Defective fetuses (%) *	0.6	3.9	2.0	0
Hydronephrosis	-	2.0**	-	-
Herniated lens and folded retina	1.1	-	-	-
Aortic arch stenosis	-	2.0	-	-
Missing right kidney and ureter	-	1.0	-	-
Cor biloculare	-	1.0	-	-
Testicular atrophy	-	1.0	-	-
Hydroencephaly	-	-	1.5	-
Spina bifida	-	-	1.5	-

 

* Based on total number of fetuses examined

** More than one defect may have appeared in 1 fetus

*** Significantly less than H2O dosed control (p<0.05)

**** The dosage was reduced to 250 mg/kg, but most died. Four survived: two were sacrificed early and two completed pregnancies. Their values were not included in statistical analyses.

 

 

MAIN STUDY:

 

There were no statistically significant differences in the gain in bodyweight nor feed consumption due to treatment among rabbits in the study. Therefore, these values are not shown. However, the group intubed with 100 mg/kg bw/d of etidronate ate the least amount of feed and gained the least amount of weight during gestation.

The overall conception rate for the 120 does used in this study was 92.5% and varied from 85% in the nontreated control group to 100% in the water-treated controls (table 2). The conception rates for the etidronate-treated groups were either 90% or 95%, so it is evident that the test material had no effect on conception nor on nidation.

As in the Pre-study, there were no significant differences in the numbers of corpora lutea, resorptions, or live fetuses. One doe, given 100 mg/kg bw/d of disodium etidronate by stomach tube, aborted at 23 days and her fetuses were dead, but this was attributed to severe respiratory disease.

The fetuses from dams given 100 mg/kg bw/d of disodium etidronate daily by gavage were significantly smaller than those from untreated control dams, but they were from slightly larger litters. Since their weights were not significantly different from the water-dosed control fetus weights, it seems likely that the reduced weight was due to normal variation and the stress of intubation on the dam.

Of the 868 rabbit fetuses examined in the main stud, 17, or less than 2%, were defective and the treated groups were not significantly different from the controls. Spina bifida and folded retina were the defects seen most often (table 2).

 

TABLE 2 (MAIN STUDY): EFFECTS OF DISODIUM ETIDRONATE ON THE REPRODUCTION AND TERATOGENY OF NEW ZEALAND RABBITS

 

	Untreated control	H2O control	25 mg/kg	50 mg/kg	100 mg/kg	100 mg/kg by gavage
No. pregnant	17	20	18	19	19	18
Conception rate	85	100	90	95	95	90
Mean corpora lutea	10.8	10.1	9.9	9.7	10.4	11.6
Mean resorptions	0.9	0.9	0.9	1.8	1.2	0.9
Mean live fetuses	7.8	8.0	8.5	7.1	8.4	9.4
Mean fetus weights (g)	32.0	30.8	30.9	30.0	28.0	27.3*
No. fetuses examined	127	155	151	134	156	145
No. with soft-tissue defects	2	4	1	1	4	4
No. with skeletal defects	0	1	0	0	0	0
Defective fetuses (%) **	1.6	3.2	0.7	0.8	2.6	2.8
Spina bifida	-	1.9	0.7	-	0.6	-
Hydroencephaly	-	0.6	-	-	0.6	-
Testicular atrophy	0.8	-	-	-	-	0.7
Cryptorchism	0.8	-	-	-	-	-
Folded retina	0.6	-	-	0.8	0.6	-
Coloboma	-	0.6	-	-	-	-
Anopthalmia	-	-	-	-	0.6***	-
Arrhinencephalia	-	-	-	-	0.6	-
Bilateral hydronephrosis	-	-	-	-	0.6	-
Gastroschisis	-	-	-	-	0.6	-
Coarctation of aorta	-	-	-	-	-	0.7

 

* Significantly less than the nontreated control group only (p<0.05)

** Based upon total number examined

*** More than one defect may have been present in a fetus

 

Applicant's summary and conclusion

Conclusions:

Disodium etidronate is not teratogenic in the rabbit under the conditions of this study. On the basis of the main study, a NOAEL of >= 100 mg/kg bw/d was derived.