(1-hydroxyethylidene)bisphosphonic acid, potassium salt

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

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read-across justification in IUCLID Section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 447 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: equivalent to 368 mg/kg bw/day active acid

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects

Remarks on result:

other: equivalent to 82.4 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects

Remarks on result:

other: equivalent to 0.5% in diet and 368 mg/kg bw/day active acid

Key result

Developmental effects observed:

no

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:

prior to GLP

Limit test:

no

Test material

Test material information

1

Reference substance name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

EC Number:

701-238-4

Molecular formula:

HEDP-2Na C2H6Na2O7P2 HEDP-3Na C2H5Na3O7P2

IUPAC Name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

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 rabbits, 5-6 months of age and about 4 kg bodyweight at 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/day 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 HEDP-2Na in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/day 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:

Gestation Day 2-16

Frequency of treatment:

Daily

Duration of test:

29 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 4

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. Mortality was observed in animals receiving 500 mg/kg bw/day after day 4 or 5 of dosing. Consequently, the 500 mg/kg bw/day dosage was reduced to 250 mg/kg bw/day. Pregnant animals were sacrificed on day 29 and examined for resorptions, corpora lutea and implantations. The foetuses 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 observed maternal toxicity from the 500 mg/kg bw/day dose of HEDP-2Na in the pre-study, doses of 25, 50 and 100 mg/kg bw/day 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 HEDP-2Na in the feed might cause different effects than when introduced by gavage, another group of rabbits was given 100 mg/kg bw/day of HEDP-2Na by a stomach tube, similarly performed as in the pre-study. Control groups, untreated and water-treated animals were included.
To reduce the possibilty of a dietary bias, all rabbits were fed ground feed which had been repelleted during both the orientation period and the 29 days of gestation.The feed consumed by 25 animals was measured for 14 days during the orientation period to establish the dietary level of test substance to be incorporated into the diet. Additionally, the feed consumed from day 2 through day 16 of pregnancy was measured for all animals to determine the actual amount of HEDP-2Na ingested as well as determine whether the stress of intubing the rabbits affected feed intake.
All animals 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 HEDP-2Na 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 foetuses were dried of amniotic fluid, sexed, carefully inspected for gross abnormalities and weighed. One-third of the foetuses were cleared, stained with Alizarin red stain (Staples and Schnell, 1964) and examined for skeletal defects. The remaining two-thirds of the foetuses 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

General toxicity (maternal animals)

Clinical signs:

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in the pre-study in animals receiving 500 mg/kg bw/day after day 4 or 5 of dosing. Four females that received only 3 doses of 500 mg/kg bw/day before having the dose reduced to 250 mg/kg bw/day survived to term. They were not included in the statistical analyses, however, their data were included so limited comparisons between the two doses could be performed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the gain in bodyweight among the rabbits. The group intubated with 100 mg/kg bw/day of HEDP-2Na consumed the least amount and gained the least amount of weight during gestation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the feed consumption among the rabbits.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, renal tubular degeneration was observed in all dams administered 250 and 500 mg/kg bw/day as well as in 20% of those dosed with 100 mg/kg bw/day. However, control animals were similarly affected (occurrence of 50%) and therefore, it was not considered as treatment-related effects.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One dam administered 100 mg/kg bw/day of HEDP-2Na by a stomach tube, aborted at 23 days and the foetuses were dead. However, this was attributed to severe respiratory disease and is considered not as a treatment-related effect.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant differences in implantations.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no significant differences in resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no significant differences in resorptions.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, New Zealand rabbits 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/day of HEDP-2Na. In the main study, the overall conception rate for the 120 rabbits was 92.5% and varied from 85% in the non-treated control group to 100% in the water-treated controls. The conception rates for the HEDP-2Na-treated groups were 90% or 95% respectively, indicating the test material had no effect on conception or on nidation.

Other effects:

no effects observed

Description (incidence and severity):

No significant differences in the numbers of corpora lutea was observed.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Basis for effect level:

other: No adverse effects observed

Remarks on result:

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

Maternal abnormalities

Abnormalities:

not specified

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

In the pre-study, the number of live foetuses was reduced in the group administered 100 mg/kg bw/day, however, this was not significant and was not observed in the main study.

Changes in sex ratio:

not examined

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

In the pre-study, no significant differences in litter weights were observed. In the main study, foetuses from dams administered 100 mg/kg bw/day of HEDP-2Na daily by gavage, were significantly smaller than those from untreated control dams. However, they were from slightly larger litters. Their weights were not significantly different from the water-dosed control foetus weights. The difference might have been caused by the stress of intubation of the dam. Additionally, the reduced weight was within the normal variation and consequently, not considered as treatment related.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

In the pre-study, no defective foetuses (18 in total) were observed in dams intubated with higher doses of the test substance. Totally, 2 of 99 foetuses from dams intubated with 100 mg/kg bw/day were defective, however, this was considered as incidental and not as treatment-related effects. Of the 868 rabbit foetuses examined in the main study, 17 (less than 2%), were defective and the treated groups were not significantly different from the controls. Spina bifida and folded retina were the most frequent occurring defects.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Totally, 30 to 40% of all foetuses in the pre-study had supernumerary ribs; most of these were bilateral. Variations in the sternebrae occurred; atrophy of the fifth sternebrae was most frequently observed. Intubation or administration of the test substance alone or in combination has affected the incidence of rib or sternal variations.

Visceral malformations:

not examined

Other effects:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

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

TABLE 1 (PRE-STUDY): EFFECTS OF INTUBATED HEDP-2Na 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.

 

TABLE 2 (MAIN STUDY): EFFECTS OF HEDP-2Na 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:

In a developmental toxicity study (reliability score 2), conducted according to OECD Test Guideline 414 and pre-GLP, HEDP (2-3Na) was administered to in concentrations of 0, 25, 50 or 100 mg/kg bw/day to 20 New Zealand rabbits. A maternal and developmental NOAEL of ≥ 100 mg/kg bw/day is concluded for rabbits.