Tetrasodium (1-hydroxyethylidene)bisphosphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

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:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Use of 2 dose levels; Sexual milestones in pups/analytical confirmation of dose not given; No sperm analyses (but conception rate of 90% suggests unaffected spermatogenesis, and OECD 416 allows omission if information is available from e.g. 90 day study)

GLP compliance:

no

Remarks:

prior to 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:

rat

Strain:

other: Charles-River

Details on test animals or test system and environmental conditions:

Weanling Charles-River rats were distributed into 5 groups, each composed of 22 females and 22 males, according to body weight and litter. They were housed in stainless steel cages with food (Ground Purina Laboratory Chow) and fresh water furnished ad libitum. 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. During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b). Pregnant females were placed in nesting cages which were furnished with a plywood liner and shredded paper.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

When appropriate, disodium etidronate was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

One male, one female placed in mating cages. Start of pregnancy determined from positive vaginal smear.

Duration of treatment / exposure:

Exposure period: continuous or Gestation Day 6-15

Frequency of treatment:

daily

Duration of test:

two-generation study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

0.5 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

22 (F0)
25 (F1b) - 20 for breeding F2 generation and 5 for histological examination

Control animals:

yes, plain diet

Details on study design:

When appropriate, disodium etidronate was mixed with the diet at levels of 0.5 and 0.1%. Both sexes were fed with the respective diets either continuously or only during the time when the females were in their sixth through fifteenth days of gestation. During the first 8 weeks, the rats were caged individually and feed consumption and body weights were recorded at weekly intervals. The rats were then paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b). Pregnancies were timed by vaginal smearing (Long and Evans, 1922), and identification of sperm in the smear designated day 0. The pregnant females were then placed in nesting cages which were furnished with a plywood liner and shredded paper.

The original females (F0) were allowed to deliver their first 2 litters, while the third litters were used for teratologic evaluation. The newborn were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly. All litters containing more than 8 pups were reduced to that number to equalize the stress on the mothers during the lactation period. After the weaning weights were recorded, all F1a pups were discarded, but 25 weanlings of each sex, from each treatment group, were selected from the F1b litters for second generation breeding stock.

20 pairs of rats from each group were bred and the remaining 5 pairs were necropsied and examined histologically. Subsequently, the first litters of the second generation (F2a) were evaluated similarly to the F1a pups, while the second litters (F2b) were used for teratologic evaluation. During the teratological phases one-half of each treatment group, selected prior to mating, were sacrificed by excessive ether inhalation on day 13 and the other half on day 21 of the gestation period.

The pregnant females were examined for number of 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).

In addition to the post weaning F1b rats, selected organs from 5 dams from each treatment group were evaluated histologically during each teratological phase.

Examinations

Maternal examinations:

Teratologic evaluation: Pregnant females were examined for number of resorptions, corpora lutea and implantations

Fetal examinations:

Teratologic evaluation: Fetuses were examined for skeletal defects, soft-tissue defects, gross abnormalities

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).

Historical control data:

In the naturally born litters, the number of pups born and weaned were comparable to those seen in previous breeding studies from this laboratory (Nolen and Alexander, 1966).

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

112 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

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

RESULTS

 

Growth, feed consumption and feed efficiency of rats fed 0.5% or 0.1% disodium etidronate from weaning to maturity were not significantly different from those of controls in either the F0 or the F1 generation, indicating that there was neither a toxic effect nor an impairment in the nutritional quality of the diet.

 

Table 1 summarizes the results of the reproductive aspects of the study involving the first 2 litters from the F0 females. There was a significant reduction in the number of live pups born to dams fed 0.5% disodium etidronateduring organogenesis in the F1a phase and an increase in the number of stillborn fetuses of F1b litters, even though these dams had a higher average number of pups than the control mothers. In the F1b phase, those dams recieving 0.1% etidronate only during pregnancy had significantly more pups than either control rats or those fed the same level continuously.

 

There were no significant differences in the preweaning mortality, weights of pups at weaning, or in the number weaned. Two litters, with an indeterminate number of pups, were stillborn in the first litters; one each in females fed 0.5 or 0.1% disodium etidronate continuously. The former had a normal second litter, but the latter did not become pregnant again.

During these 2 phases of the study, 4 of the F0 females died; 2 were in the group given 0.5% disodium etidronate continuously and 2 were from the group recieving the high level during organogenesis.The cause of death could not be determined in 2 cases. However, death of the other 2 was attributed to pneumonia in 1 and to thyroid tumor in the other.

Another of the females given 0.5% disodium etidronate during gestation died on day 25 of the third phase, 2 days after giving birth to 7 dead pups. Five more pups were found in the uterus at necropsy. One of these was exencephalic and 4 were hydroencephalic; they could not be inspected further because of decomposition.

 

Teratologic evaluation of the third litters (F1c) showed no significant treatment differences in the number of live fetuses, corpora lutea, or implantations in females sacrificed at 21 days postcoitum (table 2).

However, significantly more resorptions occurred in the control (untreated) group. In addition, 7 rats scatterd among the test and control group resorbed all their embryos.

There were no significant differences in the numbers of resorptions or of implantations in females sacrificed at 13 days postcoitum; corpora lutea were not counted in these females.

 

Only 3 malformed fetuses were found in 42 litters (0.6% incidence); 1 of these was from a mother fed 0.1% disodium etidronate continuously (left 12th rib missing) and 2 were from a mother fed 0.5% of the test material during organogenesis (hydroencephalocele in 1, sternoschisis in the other).

 

The first litters of the second generation females were smaller than the previous generation, but there were no significant differences in any of the parameters among the groups (table 3).

 

In the second litters, used for teratologic evaluation, there were no sigificant differences among corpora lutea, implantations or resorptions at 13 days nor were there significant differences between these parameters at 13 and 21 days (table 4). At 21 days the number of implantations was reduced in rats fed 0.5% disodium etidronate continuouslybut was statistically different only from those fed 0.1% of the material during organogenesis. Corpora lutea formation in rats fed 0.5% etidronate continuously and sacrificed at 21 days was significantly depressed as compared with control animals. There was a decrease in the number of live fetuses born to both groups of mothers recieving 0.5%, but the decrease was significant only in that group treated only during gestation. Second-generation fetuses were slightly heavier than those born in the first generation, but the weights were not significantly different among treatment groups.

 

More defective rats were noted in this generation, with the control group having the highest incidence. Table 5 shows the incidences and distribution of fetal anomalies in both generations. Overall, 1.2% of the 1028 fetuses examined were defective, including 2.5% of the 211 fetuses from the control group. No defect was preponderant. In addition to the abnormalities listed in table 5, some variation was seen in the incidence of extra ribs and the number of sternebrae, but the incidences of both were low and scattered randomly throughout both control and experimental groups.

In addition, neither of the tissues from the progeny nor those from the dams revealed any pathologic changes that could be ascribed to the test material.

 

 

TABLE 1: EFFECTS ON THE REPRODUCTIVE PERFORMANCE OF PARENT GENERATION (F0) RATS AND THEIR FIRST TWO LITTERS (F1a and F1b)

 

 

Group	No. pregnant		Conception rate		No. of stillborn		Av. No. born per litter		Av. No. weaned per litter		Av. weaning weight (g)
	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b	F1a	F1b
Untreated control	17	19	85	95	2	6	13.0	10.4	7.6***	7.1***	47.6	46.9
0.5% continuously	18	17	90	89.5	2	9	12.6	12.8	7.9	7.5	47.3	44.3
0.1% continuously	18	16	90	80	4	10	12.2	10.2	7.8	7.0	44.8	47.4
0.5% day 6-15 postcoitum	19	18	95	94.7	5	22	9.8*	12.7	7.1	7.6	47.1	48.3
0.1% day 6-15 postcoitum	19	18	95	90	2	15	12.7	13.5**	7.5	7.9	46.9	44.8

 

* Significantly different from control rats (p<0.05)

** Significantly different from control rats and those fed 0.1% continuously (p<0.05)

*** All litters containing more than 8 pups were reduced to that number at 4 d

 

 

TABLE 2: EFFECTS ON THE REPRODUCTION OF PARENT GENERATION (F0) RATS AND ON THEIR THIRD LITTERS (F1C)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	9	10	10	8*	9	9	9*	8*	9	7
% pregnant	90.0	100.0	100.0	88.9	90.0	90.0	100.0	88.9	90.0	70.0
No. total resorptions	1	2	1	0	1	0	1	0	1	0
Av. No. corpora lutea	-	15.3	-	14.0	-	14.3	-	12.8	-	15.5
Av. No. implantations	13.0	13.4	14.3	14.4	12.8	13.6	16.3	13.4	13.3	14.4
Av. No. resorptions	2.0	3.9**	0.3	0.0	1.3	0.0	1.8	0.0	0.6	0.0
Av. No. live fetuses	-	11.6	-	14.4	-	14.3	-	13.4	-	14.4
No. dead fetuses	-	0	-	0	-	0	-	1	-	0
No. fetuses abnormalities	-	0	-	0	-	1	-	2	-	0
Av. weight fetuses (g)	-	3.5	-	3.5	-	3.9	-	3.5	-	3.7

 

* Deaths earlier had reduced these groups in number

** Significantly different from other females sacrificed at 21 d (p<0.05)

 

 

TABLE 3: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR FIRST LITTERS (F2a)

 

	Control	0.5% continuously	0.1% continuously	0.5% days 6-15 postcoitum	0.1% days 6-15 postcoitum
No. pregant	19	15	20	19	18
% pregnant	95.0	75.0	100.0	95.0	90.0
Av. No. born live per litter	10.2	9.1	8.9	9.1	10.3
Av. pup weight at 4d (g)	9.7	9.5	10.4	10.1	9.6
Av. No. weaned per litter*	7.1	7.4	6.7	7.0	7.4
Av. weight of pups  at weaning (g)	43.0	41.7	46.0	45.5	42.9

 

* Litters containing more than 8 pups were reduced to that number at 4 d

 

 

 

TABLE 4: EFFECTS ON THE REPRODUCTION OF SECOND GENERATION PARENTS (F1b) AND ON THEIR SECOND LITTERS (F2b)

 

Effect	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d	13 d	21 d
No. pregant	10	10	10	7	10	9	9	9	10	10
% pregnant	100.0	100.0	100.0	70.0	100.0	90.0	90.0	90.0	100.0	100.0
No. total resorptions	0	1	1	0	1	0	2	1	1	0
Av. No. corpora lutea	13.9	14.2	13.6	11.9*	13.6	13.6	13.4	13.4	13.2	13.8
Av. No. implantations	14.3	13.1	12.0	10.7**	13.0	13.4	13.3	11.2	13.0	13.5
Av. No. resorptions	1.2	1.4	0.9	0.9	1.8	0.9	1.9	3.8	1.3	0.9
Av. No. live fetuses	-	13.0	-	9.9	-	12.4	-	9.1***	-	12.6
No. dead fetuses	-	0	-	0	-	0	-	0	-	0
No. fetuses abnormalities	-	5	-	1	-	1	-	0	-	3
Av. weight fetuses (g)	-	4.8	-	5.4	-	5.4	-	4.9	-	5.2

 

*Significantly less than control rats at 21 d (p<0.05)

** Significantly less than rats fed 0.1% days 6-15 postcoitum (p<0.05)

*** Significantly less than control rats (p<0.05)

 

 

TABLE 5: INCIDENCES AND DISTRIBUTION OF FETAL ABNORMALITIES OBSERVED IN RATS

 

	Control		0.5% continuously		0.1% continuously		0.5% days 6-15 postcoitum		0.1% days 6-15 postcoitum
	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b	F1c	F2b
No. litters	7	9	8	7	9	9	7	8	7	10
No. fetuses examined	93	118	115	69	122	113	94	74	102	128
No. fetuses examined for skeletal defects	31	37	38	22	39	38	30	24	33	43
No. fetuses examined for soft-tissue defects	62	81	77	47	83	75	64	50	69	85
Fetuses with gross abnormalities (%)	0	2.5*	0	0	0	0	1.1	0	0	0
Fetuses with skeletal defects (%)	0	0	0	0	2.5~	0	3.3	0	0	0
Fetuses with soft-tissue defects (%)	0	5.0#	0	2.1	0	1.3	1.1	0	0	3.5
Facial dysgenesis	-	1.7	-	-	-	-	-	-	-	-
Hematoma	-	0.8	-	-	-	-	-	-	-	-
Hydronephrosis	-	2.5	-	-	-	1.3	-	-	-	1.2
Double aorta	-	1.2	-	-	-	-	-	-	-	-
Cryptorchism	-	-	-	2.1	-	-	-	-	-	-
Hydro-encephalocele	-	-	-	-	-	-	1.1	-	-	-
Ascites	-	-	-	-	-	-	-	-	-	1.2
Hyperplastic testicle	-	1.2	-	-	-	-	-	-	-	-
Abdominal hemorrhaging	-	-	-	-	-	-	-	-	-	1.2
Missing rib, L 12th	-	-	-	-	2.5	-	-	-	-	-
Sternoschisis	-	-	-	-	-	-	1.1	-	-	-

 

* Percentages are based upon total number of fetuses examined

~ Percentages are based upon number cleared and stained

# Percentages are based upon number of animals examined for soft-tissue effects

Applicant's summary and conclusion

Conclusions:

In a two-generation study with rats, no treatment-related malformations or developmental variations were noted at any exposure level.

Executive summary:

A two-generation feeding study in rats was conducted with disodium etidronate to evaluate the effects on reproduction functions and embryogeny. The doses in the diet were 0, 0.1 or 0.5% (equivalent to 0, or appr.112 or appr. 447 mg/kg bw/d) and were given either continuously to both sexes or to females only on GD 6-15.

The parent generation (F0) was allowed to deliver two litters (F1a and F1b), while the third (F1c) was used for teratologic evaluation. Reproductive performance parameters (number pregnant, conception rate, number of stillborn, average number born per litter, average number weaned per litter, average weaning weight) were evaluated for the F1a and F1b generation. From each of the F1b dose groups, 5 pairs were necropsied and examined histologically and 20 pairs were selected for the second generation breeding stock. The first litters (F2a) of the second generation were handled according to F1a and F1b, while the second litter (F2b) was like F1c used for teratologic evaluation.

With regard to the F0 generation, growth, feed consumption and pregnancy rate were comparable between the control and the dose groups. On the basis of the overall conception rate of 90%, the authors concluded that spermatogenesis and nidation were not affected by the intake of disodium etidronate.

With regard to fetotoxicity, the following effects were observed in the litters:

1) No effects in the F1c (third litter of the F0 rats) and F2a (first litter of F1b parents) generation,

2) Not significantly increased number of stillborn in the F1b generation (only in the highest dose groups of females dosed on GD 6-15)

3) Average number of corpora lutea significantly decreased (11.9) compared to control (14.2) (only in the F2b generation in the highest dose group, only in continuously dosed females, only after 21 but not after 13 days)

4) Significantly reduced average number of live fetuses (9.1) compared to control (13.0) (only in the F2b generation in the highest dose group, only in the females dosed on GD 6-15, but not in continuously dosed dams)

The authors concluded that no general toxic effect was observed which could be traced back to the intake of the test substance. Maternal toxicity is often observed in fertility and/or developmental toxicity assays at dose levels lower than those obtained from general repeated dose toxicity studies. Considering that the doses applied in the study surpassed the NOAEL deduced from repeated dose toxicity studies (78 mg/kg bw/d for adult animals and 41 mg/kg bw/d for juvenile animals) by far, it is likely the maternal toxic effects in form of perturbation in haematological parameters and anaemia occurred, but were not in the scope of the investigation. Another sign of maternal toxicity is the occurrence of five deaths of dams in the highest dose groups. While one death was attributed to the occurrence of pneumonia and another to a thyroid tumor, the cause of death could not be determined in the three other cases. In one of these three cases, the female gave birth to 7 dead pups 2 days before her death. It remains unclear whether these 7 dead pups were counted to the overall number of stillborn or not. Altogether, the deaths of dams only in the highest dose groups and the assumption that haematological effects occurred, but were not detected, puts the effects observed in the litters into relation. In addition, these effects gave no consistent picture throughout the dose groups. For example, the number of corpora lutea was affected in the continuously dosed animals, but not in those fed from GD 6-15, while the number of live fetuses was decreased only in the latter group.

The total number of fetuses examined for developmental effects was 1028 with the highest number of fetal abnormalities occuring in the F2b control group. In light that the incidence of defective pups in all dose groups did not differ from those in the control, the authors concluded that disodium etidronate was not teratogenic to rats at the dose levels tested.

In conclusion, an impairment of reproductive function was not observed in male or female rats at relatively high dose levels. From the two-generation study, no treatment-related malformations or developmental variations were noted at any exposure level. Intrauterine parameters (mean numbers of corpora lutea, implantation sites, resorptions, viable fetuses, and mean fetal weights) were unaffected by treatment at an exposure level of 112 mg/kg bw/d (1.4 times higher than the NOAEL of 78 mg/kg bw/d for adult animals). The occurrence of fetotoxic effects can not be excluded at high doses which surpass the general systemic NOAEL in rats by a factor of 6 and are therefore likely to induce maternal toxicity.