Tetrasodium (1-hydroxyethylidene)bisphosphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The review of six studies/publications for sodium salts of HEDP and of three studies for other structurally homologues gave no indication for a potential of phosphonates to affect fertility in rats, dogs or mice.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive 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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Species:

rat

Strain:

other: Charles-River

Sex:

male/female

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.

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.
Females have been determined to be in proestrus by vaginal smearing and continuous exposure started "from weaning", i.e. in week 5.

Details on mating procedure:

After 8 weeks, the rats were paired and placed into mating cages. This procedure was repeated for the breeders in the second generation (F1b).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: continuous

Frequency of treatment:

daily

Details on study schedule:

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.

Twenty (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.

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet

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

Parental animals: Observations and examinations:

Feed consumption and body weights were recorded at weekly intervals.
Evaluation of conception rate, no. of stillborn, no. of born per litter, no. of weaned per litter

Oestrous cyclicity (parental animals):

Pregnancies were timed by vaginal smearing (Long and Evans, 1922)

Litter observations:

All newborn were counted at birth, but not handled until they were 4 days old. They were then weighed, sexed and inspected grossly.
- F1a: discarded
- F1b: 5 per sex were necropsied and examined histologically.
- F1c: teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations
- F2a: discarded
- F2b: teratologic evaluation (one-half of each treatment group on day 13, the other half on day 21 of gestation period). The pregnant females were examined for number of resorptions, corpora lutea and implantations.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

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).

Dose descriptor:

NOAEL

Effect level:

112 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

112 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Reproductive effects observed:

not specified

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

Conclusions:

An impairment of reproductive function was not observed in male or female rats at relatively high dose levels. The overall conception rate of 90% shows that the compound did not interfere with the spermatogenesis nor nidation, throughout the testing period of 2 generations.

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. Females have been determined to be in proestrus by vaginal smearing andcontinuous exposure started "from weaning", i.e. in week 5.

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.

Reference 2

Endpoint:

fertility, other

Type of information:

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

Adequacy of study:

key study

Study period:

19.07.1976 to 17.07.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restriction was that only 40 instead of 50 animals were used.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies). Study conducted prior to adoption of OECD guideline.

Deviations:

yes

Remarks:

Only 40 animals used (50 in guideline)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anglia Laboratory animals, UK
- Age at study initiation: No data
- Weight at study initiation: 75-90 g
- Fasting period before study: No
- Housing: Five per cage in suspended cages with wire-mesh floors.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Six days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2
- Humidity (%): 50 ±5
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 19.07.76 To: 17.07.78

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Powdered laboratory rat food: Spratts Laboratory Diet 2.
- Storage temperature of food: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were sent to the Sponsor for analysis of diets fed during week 30 and at approximately three month intervals thereafter. No further details provided.

Duration of treatment / exposure:

104 w

Frequency of treatment:

continuous

Details on study schedule:

none

Dose / conc.:

500 ppm

Remarks:

Males: 19 mg/kg bw/day (expressed in terms of material as supplied)
Females: 24 mg/kg bw/day (expressed in terms of material as supplied)

Dose / conc.:

2 000 ppm

Remarks:

Males: 78 mg/kg bw/day (expressed in terms of material as supplied)
Females: 96 mg/kg bw/day (expressed in terms of material as supplied)

Dose / conc.:

10 000 ppm

Remarks:

Males: 384 mg/kg bw/day (expressed in terms of material as supplied)
Females: 493 mg/kg bw/day (expressed in terms of material as supplied)

No. of animals per sex per dose:

40

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Based on body weight
- Rationale for selecting satellite groups: Used to provide blood and urine samples during the first 26 weeks of the study, and were therefore subjected to the stresses of collecting these samples. Hence the main group animals were not subjected to these stressors until the end of the 102 week exposure period.
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale (if not random): No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first eight weeks, and two-weekly thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, mean weekly intake calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: During week 4 for a 5-day period for each cage in control and high dose level main groups. During weeks 11 and 26 for a 5-day period for each cage of all main groups.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During weeks 52 and 104
- Dose groups that were examined: Examined in all surviving males and female rats from control and top dose groups.


HAEMATOLOGY: Yes
- Time schedule for collection of blood and parameters measured: Weeks 0 and 5 from 10 males and females from Control and highest dose; Week 12 from 10 males and females in all groups; Weeks 25 and 102 from 10 males and females from control, mid and highest dose groups: packed cell volume, haemoglobin, red cell count, mean corpuscular haemoglobin concentration and mean cell volume, total white cell count and differential count. Platelet count and thrombotest were conducted in weeks 12, 25 and 103 only. A visual estimation of red cell count and RBC osmotic fragility was conducted on the blood from high dose satellite group animals immediately prior to post-mortem.
- Anaesthetic used for blood collection: Yes (not identified)
- Animals fasted: Yes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 5, 12, 25 and 102: 5 males and 5 females from control and 10000ppm; plasma urea, plasma glucose, total serum proteins, serum alkaline phophatase, serum glutamic pyruvic transaminase, sodium, potassium, calcium, inorganic phosphorus, serum creatinine. Week 7: 5 males from control, 2000ppm and 10000ppm for glucose and serum alkaline phosphatase. Week 12: 5 males from 500ppm and 2000ppm for serum alkaline phosphatase and serum glutamic pyruvic transaminase. Week 13: 5 females from all groups - plasma glucose. Week 25: 5 males from 2000ppm group for serum alkaline phosphatase and 5 females from 2000ppm group for plasma glucose.
- Animals fasted: Yes


URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 6, 25 and 102: 5 males and 5 females from control and highest dose: pH, specific gravity, protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, haemoglobin, microscopy of spun deposits, urinary calcium, urinary phosphorus. Week 7: samples collected from 5 males and 5 females from all groups for estimation of pH, specific gravity and volume. During week 12: individual overnight urine samples from 5 males and 5 females from all groups. Urinary hydroxyproline measured in control and top dose at week 26.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No

Statistics:

One way analysis was performed on each parameter and treated groups compared with control using Student's t- test. Used for organ weight data, urinalysis, haematology, blood chemistry and bodyweights, food consumption, water consumption.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

CLINICAL SIGNS AND MORTALITY: No treatment related effects. Survivors at study termination: Males - 14 (control), 16 (500ppm), 23 (2000ppm) and 20 (10000ppm); Females - 14 (controls), 22 (500ppm), 19 (2000ppm) and 19 (10000ppm). At 10000ppm severe pallor of skin was observed from week 6, regressed from week 35 and was back to normal by week 68. At 2000ppm slight pallor of skin was observed from weeks 6-35, regressed and returned to normal by week 52. There were no clinical signs of toxicity in the 500 ppm group.


BODY WEIGHT AND WEIGHT GAIN: In the 10000ppm group lower bodyweight gain in males during first 13 weeks only, and in females during first 12 weeks, and from weeks 26-52, were observed. However overall no consistent intergroup differences.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No consistent intergroup differences suggestive of reaction to treatment. However, doses received in the main study were much lower than those in thhe associated satellite study over the first 13 weeks of the two year study, due to higher food intake as a function of bodyweight in the younger animals.


FOOD EFFICIENCY: There were no consistent differences found between treated and control animals.


WATER CONSUMPTION: Slight differences noted which were attributed to slight differences in food consumption between groups.


OPHTHALMOSCOPIC EXAMINATION: No treatment-related effects.


HAEMATOLOGY: Perturbations were recorded at early times but there were no treatment-related effects that persisted until 104 weeks. In the animals treated with 10000ppm, lower values relating to red cell parameters in both sexes were observed during weeks 5, 7, 12.  Lower values for packed cell volume and haemoglobin concentrations and an increased red cell count in both sexes during week 25 were reported. Microcytosis noted at week 25 and 26. At week 5 all animals receiving 10000ppm showed slight polychromasia and/or hypochromasia. These observations were extended to the lower group at week 7 and showed lower red cell values in 2000ppm and 10000ppm group males. All rats treated with 10000ppm had many abnormalities of the red blood cells. Examination of blood films from males receiving 2000ppm revealed only slight to moderate anisocytosis, polychromasia and hypochromasis in some of the rats. At week 12 anaemia was still noted among males and females receiving 10000ppm and among males only receiving 2000ppm,. Many blood cell abnormalities noted in 10000ppm male group. By 25 weeks the values relating to red cell parameters were similar to controls for the 2000ppm group and the packed cell volume and haemoglobin concentration were only marginally lower in the 10000ppm group. However the red cell count for 10000ppm group was higher than the control. An increase in microcytosis and presence of giant platelets was seen in some of the 10000ppm group animals.  Red cell fragility examinations conducted at week 26 indicated the red cells from animals treated with 10000ppm could shrink in the presence of normal plasma.  It was considered that the microcytosis observed in association with the anaemia in rats receiving 100000ppm could be due to an effect in the circulation rather than at source.  No microcytosis was observed in observations conducted after 26 weeks. Increased neutrophil and lymphocyte counts in both sexes in the 10000ppm group during weeks 6 and 7 and in the 2000ppm male group when the observations were extended to the lower group at week 7.  Although marginally higher lymphocyte count was seen in 2000ppm and 500ppm females during week 7 these were not statistically significant. No differences were seen at later sampling times. There was a marginally higher platelet count in high dose group males during weeks 12 and 25.  


CLINICAL CHEMISTRY: At 10000ppm higher serum alkaline phosphatase in males at week 5, 7, 12 was reported. There were no effects at lower doses.  Although there were some inter-group differences, none were consistent, outside normal ranges or considered to be of toxicological relevance.


URINALYSIS: Marginally increased urine volume were found in males of all groups at week 7, but not at other sampling times. Some individual perturbations of pH were observed, but not considered to be of toxicological significance.  At 25 weeks the highest dose groups had higher levels of calcium and inorganic phosphorous, which could be attributed to the chelating activity of the test compound. This effect was not seen at later sample times.


ORGAN WEIGHTS: At 10000ppm lower liver weights in both sexes and lower kidney weights in males at 26 weeks, but not 104 weeks, was reported. Lower liver weights were recorded for males at 26 weeks, but not 104 weeks, in the 2000 ppm group. In the 500 ppm group lower liver weights among females at 26 weeks, but not 104 weeks, were reported.


GROSS PATHOLOGY: No treatment-related findings.


HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related changes were observed only in the spleen.  These consisted of a lack of iron in a proportion of male rats receiving 2000ppm and 10000ppm, and in female rats receiving 10000ppm at 26 weeks but not 104 weeks.  No evidence of treatment-related effects was found in the 500 ppm group at 26 or 104 weeks. There were no treatment-related changes relating to non-neoplastic lesions and those observed were considered normal for this strain and age.


HISTOPATHOLOGY: NEOPLASTIC: No increased incidence of neoplastic lesions was observed in treated groups at 104 weeks.

Dose descriptor:

NOAEL

Effect level:

>= 384 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: only as far as effects on reproductive organs are considered

Dose descriptor:

NOAEL

Effect level:

>= 493 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: only as far as effects on reproductive organs are considered

Remarks on result:

other: F0 were not mated

Reproductive effects observed:

not specified

 

	Group
No. of rats with:	1♂		2♂		3♂		4♂		1♀		2♀		3♀		4♀
	D	T	D	T	D	T	D	T	D	T	D	T	D	T	D	T
REPRODUCTIVE SYSTEM Testes Arrest spermatogenesis / atrophy / dilation of tubules / peri-arteritis	7	2	12	2	8	6	4	6
Dystrophic mineralisation of blood vessels		1			3	1	1
Miscellaneous including congestion, haemorrhage, oedema, inflammation in the epididymis, spermatocele in the epididymis				1	2		1
Prostate Prostatis	3	1	2
Low epithelium / hyperplasia of epithelium	2
Seminal vesicles Lack of secretion within acini	3	2				1
Distended acini / low epithelium			1					1
Inflammatory cell infiltration / abscess		1	2		2

 

D = Rats dying or killed during the study                      T = Rats killed at termination of study

  

 

	Group
No. of rats with:	1♂		2♂		3♂		4♂		1♀		2♀		3♀		4♀
	D	T	D	T	D	T	D	T	D	T	D	T	D	T	D	T
Ovaries Lack of atretic / cystic follicles / cysts									3	1	1	4	3	1	2	5
Lack of corpora lutea									1			1		7	1	5
Corpora lutea +/- haemorrhage										2					1
Miscellaneous including pigment deposition cyst in oviduct										1			1	1
Uterus Congestion of blood vessels / mononuclear cell aggregations									1			1		2
Dilation lumen / endometrial gland												1	1	3	1	1
Hyperplasia / squamous metaplasia														1		1
Miscellaneous including intussusception infarction, necrosis in lumen, polyp									1			1		1	1	1
Mammary gland Hyperplasia / galactocele / dilated duct +/- increased connective tissue / fibrosis			4	1	3	2	0		8	7	2	12	4	9	3	13
Miscellaneous including pigmented macrophages inlammatory cell infiltration							1								1

 

D = Rats dying or killed during the study                      T = Rats killed at termination of study

Conclusions:

Macroscopic and microscopic examination revealed any abnormalities in gonads (ovaries & testes) and accessory reproductive organs (uterus, cervix, mammary, endometrial & mammary glands, testes, preputial gland, prostate, & seminal vesicles).
The NOAEL for effects on reproductive organs in this study is therefore >= 384 mg/kg/day in male rats and >= 493 mg/kg/day in female rats.

Executive summary:

In this well-performed study, in which sodium salt of Complexing Agent - Henkel 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate) was administered to Sprague-Dawley-derived rats (40/sex/dose) in the diet for 104 weeks, at dietary concentrations of 500, 2000, 10000 ppm (equivalent to 19, 78 and 384 mg/kg/day for males, and 24, 96, 493 mg/kg/day for females). The animals were observed for mortality, clinical signs of toxicity, food and water consumption, ophthalmoscopy, and gross and microscopic examinations. There was no evidence of neoplastic potential, or other chronic toxicity effects for disodium etidronate apart from the perturbations of haematological parameters that were observed in the highest dose groups (discussed in Section 7.5.1).

Regarding reproductive organs the weights of testes, seminal vesicles, and prostate of the treated animals were comparable with those of the control group. Histology of the testes, prostate and seminal vesicles from treated rats showed parameters like spermatogenesis, atrophy, dilation of tubules, or peri-arteritis within the normal range. No histological or weight differences were found between ovaries, uterus, or mammary (gland) in treated and untreated rats. The collective results indicate that the treatment with disodium etidronate induces no suppression of gonadal functions.

The NOAEL for effects on reproductive organs in this study is therefore >= 384 mg/kg/day in male rats and >= 493 mg/kg/day in female rats.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

The in-life phase of the study was conducted between 13 June 2012 (first day of treatment) and 04 August 2012 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 306 to 351g, the females weighed 189 to 219g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C
- Humidity (%): The relative humidity control was set to achieve target values of 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water. Formulations were prepared fortnightly and stored at approximately 4ºC in the dark.

All formulation were adjusted for 10.95% water content. All dose levels of expressed in terms of Active Ingredient

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken and analysed for concentration of test item.

The concentration of test item in the test item formulations was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using an external standard technique.

The results indicate that the prepared formulations were within ± 3% of the nominal concentration.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation).

Frequency of treatment:

The test item was administered daily.

Details on study schedule:

Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

active ingredient

Dose / conc.:

350 mg/kg bw/day (actual dose received)

Remarks:

active ingredient

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

active ingredient

No. of animals per sex per dose:

10 males and 10 females per dose (including control)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were chosen based on the results of a 7-day range-finding study.

Positive control:

No positive control.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
- Motor Activity
- Forelimb/Hindlimb Grip Strength
- Sensory Reactivity


BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

REPRODUCTIVE SCREENING:
Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

Oestrous cyclicity (parental animals):

During mating a vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

Sperm parameters (parental animals):

During histopathology, the male testes and epididymides were examined.

Litter observations:

Litter Data:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

All tissues were despatched to the histology processing Test Site for processing.
Microscopic examination was conducted by the Study Pathologist

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.

Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Reproductive indices:

Mating Performance and Fertility:

The following parameters were calculated from the individual data during the mating period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated ÷ Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females ÷ Number of animals mated) x 100

Gestation and Parturition Data :
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring ÷ Number of pregnant females) x 100

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY:
There were no unscheduled deaths.

CLINICAL OBSERVATIONS:
No clinical signs of toxicity were detected in any treated animal.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

FUNCTIONAL PERFORMACE TEST:
There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODYWEIGHT:
There were no toxicologically significant effects detected in body weight development.

FOOD CONSUMPTION:
No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

WATER CONSUMPTION:
Daily visual inspection of water bottles did not reveal any significant intergroup differences.

REPRODUCTIVE PERFORMANCE:
Mating:
There were no treatment-related effects on mating performance.

Fertility:
There were no treatment-related effects on fertility.
One female treated with 100 mg/kg bw/day A.I. was non-pregnant. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of this missing pregnancy.

Gestation Length:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 23½ days.

HAEMATOLOGY:
There were no toxicologically significant effects detected in the haematological parameters examined.

BLOOD CHEMISTRY:
There were no toxicologically significant effects detected in the blood chemical parameters examined.


PATHOLOGY:
NECROPSY:
Adults:
No toxicologically significant macroscopic abnormalities were detected.

ORGAN WEIGHTS:
No toxicologically significant effects were detected in the organ weights measured.

HISTOPATHOLOGY:
No treatment related microscopic findings were detected.

Dose descriptor:

NOEL

Remarks:

(reproductive toxicity)

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related effects detected in the reproductive parameters observed.

Dose descriptor:

NOEL

Remarks:

(systemic toxicity)

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: The oral administration of test item to rats by gavage, at dose levels of 100, 350 and 1000 mg/kg bw/day A.I., did not result in any toxicologically significant effects.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Litter Responses:
In total nine females from control and 100 mg/kg bw/day A.I. dose groups and ten females from 350 and 1000 mg/kg bw/day A.I. dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Pre and post implantation losses were increased in all treated groups when compared to control females. Statistical analysis of the data did not reveal any significant intergroup differences and a true dose related response was not evident. The number of corpora lutea, implantation sites and number of offspring born were also comparable to control females therefore the intergroup differences were considered not to be of toxicological importance.

Offspring Growth and Development:
There were no toxicologically significant effects detected.

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Pathology:
Necropsy:
Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Reproductive effects observed:

not specified

Conclusions:

The oral administration of [[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt to rats by gavage, at dose levels of 100, 350 and 1000 mg/kg bw/day A.I., did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day A.I., equivalent to 800 mg/kg bw/day active acid.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day A.I., equivalent to 800 mg/kg bw/day active acid.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Ha:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 1000 mg/kg bw/day A.I. (adjusting for 10.95% water content). A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Adult Responses:

Mortality. There were no unscheduled deaths.

Clinical Observations. No clinical signs of toxicity were detected in any treated animal.

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Body Weight. There were no toxicologically significant effects detected in body weight development.

Food Consumption. No adverse effect on food consumption or food efficiency was detected in treated animals.

Water Consumption. No adverse effect on water consumption was detected.

 

Reproductive Performance:

Mating. There were no treatment-related effects on mating for treated animals.

Fertility. There were no treatment-related effects on fertility.

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

 

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development.Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

 

Laboratory Investigations:

Haematology. There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry. There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Pathology:

Necropsy. No toxicologically significant macroscopic abnormalities were detected.

Organ Weights. No toxicologically significant effects were detected in the organ weights measured.

Histopathology. No treatment related microscopic abnormalities were detected.

 

Conclusion.

The oral administration of [[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium saltto rats by gavage, at dose levels of 100, 350 and 1000 mg/kg bw/day A.I., did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day A.I.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day A.I.

Reference 4

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

12.09. 1977 to 19.06.1978

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

Conducted prior to adoption of OECD test guideline.

Deviations:

yes

Remarks:

No analytical confirmation of exposure, no pre-mating exposure, no assessment of oestrus cycle or sperm analyses, no evaluation of sexual milestones in pup. Males treated only as F1 and F2 generation.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Females: Blue Spruce Farms. Males: BioDynamics Inc. in-house Long Evans breeding colony.
- Age at study initiation: (P) x 15-16 wks; (F1) x 3 wks
- Weight at study initiation (means): Females approximately 240 g. Males not weighed.
- Fasting period before study: No
- Housing: Individually in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12.09. 1977 to 19.06.1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow
- Storage temperature of food: No data

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: nightly until signs of mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually in elevated stainless steel cages.
- Any other deviations from standard protocol: Dosing not started until Day 0 of gestation.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Test substance administration to F0 females was initiated on gestation Day 0 and continued throughout gestation and lactation, and for F1males and females through the F2a and f2b litters.

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until one week after selection from the F1 litters.
- Selection of parents from F1 generation when pups had reached maturity.

Dose / conc.:

300 ppm

Remarks:

nominal in diet

Dose / conc.:

1 000 ppm

Remarks:

nominal in diet

Dose / conc.:

3 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

F0 females: 20.
F1 Parents: females - 20; males - 10.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random.
- F1 offspring were separated from siblings seven days after weaning of the last litter and were randomly selected to continue as future parents (f1). More offspring than needed were selected (12 males and 24 females) for the growth period to insure the required number of adults (10 males and 20 females) necessary for mating. Following pup selection, remaining offspring and F0 females were sacrificed and discarded after gross external and internal examinations.
F1 animals were raised to maturity and mated to produce the F2a litters. F2a pups were sacrificed, necropsied and discarded at weaning. All F1 females were remated after a rest period of at least 14 days to produce the F2b litters. F2b pups were sacrificed and necropsied at weaning. Following completion of the F2b sacrifice, all F1 parents were sacrificed, necropsied and selected tissues preserved.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and gross signs of toxicity in F0 and F1: twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly (F0 and F1)


BODY WEIGHT: Yes
- Time schedule for examinations: Males and nonpregnant females (F1): weekly in growth and rest periods of F1 generation. Pregnant females (F0 and F1): Days 0, 6, 15 and 20 of gestation. Lactation females (F0 and F1): Days 0, 4, 14 and 21 of lactation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Males and nonpregnant females: weekly for growth and rest periods of F1 generation.
-Test substance consumption was calculated from body weight and food consumption values: Males and nonpregnant females: weekly during growth and rest periods of F1 generation.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Not examined as dosing in F0 dams started on Day 0 gestation, F0 males not dosed.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: No data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED:
Gestation period: On day 19 of gestation, dams were provided with nesting material and were examined daily for signs of parturition.
Lactation period (Day 0 is day on which all pups had been delivered): General appearance of pups and presence of dead pups examined daily in all litters (F1, F2a and F2b). LIve, dead and missing pups were recorded on Days 0, 1, 4 (pre-cull and post-cull), 14 and 21 of lactation in all litters (F1, F2a and F2b). Live pups were weighed as a litter on Days 0, 4 (pre-cull) and 21 (calculated from individual weights) of lactation for all litters, and individually on Day 21 of lactation for all litters.
Litter Data and Observations: The number of each sex per litter was determined on Days 0 and 4 (pre- and post-cull) of lactation for all litters. Individual sex determination on Day 21 of lactation for all litters.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
F0 generation: All females sacrificed after weaning of F1 offspring, and necropsy performed.
F1 generation: All surviving males and females sacrificed after weaning and necropsy of the last F2b litter. Necropsy performed, and selected organs weighed and tissues preserved.
Extra F1 males and females: sacrifice at initiation of mating, necropsy performed and discarded.
Dead and moribund dams: Necropsy performed. Uterine contents examined and presence of implantation sites and/or scars recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were weighed and/or prepared for microscopic examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning. Necropsy performed including internal sex determination peformed, then carcassed discarded.
- F2a: necrospy included internal sex determination performed. All offspring where then discarded.
- F2b: necrospy included internal sex determination performed. Selected tissues were preserved from ten randomly chosen males and females from all groups.
F2b found dead after Day 14 of lactation: necropsy including internal sex determination performed and discarded.
Culled F1, F2a and F2b: sacrifice on Day 4 of lactation. Necropsy including internal sex determination performed and discarded.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dead pups: sex was determined and pups checked for presence of milk in their stomach and discarded.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were preserved for F2b offspring.

Statistics:

Body weight, body weight gain, food consumption and litter examination data, organ weights, organ/body weight and organ/brain weight ratios were analysed. Group I and Group II (control groups) were compared to each other. If no statistically significant differences, the control groups were combined and compared against test groups. If controls differed, individual control/test comparisons conducted - continuous and discrete data (body weight, food intake, organ weight): Dunnett's test.  - gestation length: F-test and Student's T-test (Cochran's approximation). - offspring data: Chi-square. Incidence data for control Group II was compared to control Group I and incidence data for each test substance treated group was compared to each control group.

Reproductive indices:

F0 females: Pregnancy rates, gestation length, percentage of mothers that weaned litters.
F1 females: Mating and fertility indices, pregnancy rates, parturition indices, gestation length, percentage of mothers that weaned litters.

Offspring viability indices:

F1 litters: Mean numbers of live and dead pups at birth, pup survival (at representative intervals through lactation), body w eight, sex distribution.
F2 litters: Mean numbers of live and dead pups at birth, pup survival (at representative intervals through lactation), body w eight, sex distribution.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No parental animals died or showed clinical signs of toxicity.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There were no effects on body weight/body weight gain.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): There are no comments on this, but as food consumption was not affected, test substance intake should also have not been affected.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No effects. A statistically significant reduction in mean gestation length for F2a animals from the 1000 ppm group (22.1 d) versus the pooled F2a controls (22.4 d) was considered spurious by the authors. Also, the lower pregnancy rate recorded in the F1/F2a mating (not significant) was not replicated in the second mating (F1/2b).


ORGAN WEIGHTS (PARENTAL ANIMALS): No effects found.


GROSS PATHOLOGY (PARENTAL ANIMALS): No abnormal findings.


HISTOPATHOLOGY (PARENTAL ANIMALS): No abnormal findings.

Dose descriptor:

NOAEL

Effect level:

ca. 882 mg/kg bw/day

Sex:

male

Basis for effect level:

other: 3000 ppm

Dose descriptor:

NOAEL

Effect level:

ca. 936 mg/kg bw/day

Sex:

female

Basis for effect level:

other: 3000 ppm

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING): The mean numbers of live and dead pups at birth (F1) were considered comparable between the controls and 300 and 1000 ppm groups. In the high dose group there was a decrease in the mean numbers of live pups with a corresponding increase in the mean number of dead pups observed at birth. The differences were not statistically significant compared with the combined control values. The 24 hour and four day survival indices for the treated groups were slightly lower than the control values, and some statistically significant differences from a control group were observed. There was no dose-response relationship so it was concluded that the difference were not treatment-related. There was a slight increase in the mean number of dead pups at birth in the F2a litters at the high dose. However, the increase was attributed to one female that had five dead pups. Pup survival indices were comparable between the groups for the F2 generation.


CLINICAL SIGNS (OFFSPRING): There were no clinical signs of toxicity.


BODY WEIGHT (OFFSPRING): In the highest dose group (F1) there was a non-statistically significant reduction in mean pup weights at birth. There was a slightly lower pup birth weight in mid and high dose groups, but the difference was only statistically significant in the highest dose group compared with the combined controls. There was no difference at other time points or in the F2b generation.


GROSS PATHOLOGY (OFFSPRING): No abnormal findings.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 294 mg/kg bw/day

Sex:

male

Basis for effect level:

other: 1000 ppm

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 312 mg/kg bw/day

Sex:

female

Basis for effect level:

other: 1000 ppm

Reproductive effects observed:

not specified

Table 2 Summary of Gestation length, litter survival, growth of offspring and weaning sex ratio data.

Group (ppm)	Mean gestation period (days)	Mean No.		Mean No. weaned/litter	Litters weaned		Mean live offspring weights (g)				Day 21 sex ratio (M/F)
		Live	Dead		No	%	Day 0	Day 4^a	Day 14	Day 21	No.	Ratio^b
F0 to F1
I/0	22.1	12.3	0.1	9.8	18/18	100	5.9	9.4	27.3	40.8	86/90	0.96
II/0	22.1	11	0.6	8.9	17/18	94.4	5.9	9.3	27.0	41.5	71/81	0.88
I+II	22.1	11.6	0.3	9.4			5.9	9.3	27.2	41.1
III/300	21.9	11.3	0.8	9.5	17/18	94.4	5.9	9.6	27.9	41.3	81/81	1.00
IV/1000	21.9	10.9	0.6	8.7	17/17	100	6.1	9.8	27.3	40.4	65/83	0.78
V/3000	22.3	9.2	1.9	7.9	17/19	89.5	5.7	8.5	26.6	40.8	64/70	0.91
F1 to F2a
I/0	22.4	10.9	0.1	8.7	13/14	92.9	6.3	9.7	26.6	35.4	55/58	0.95
II/0	22.4	10.5	0.3	9.1	14/15	93.3	6.1	10.1	25.5	35.3	64/63	1.02
I+II	22.4	10.7	0.2	8.9			6.2	9.9	26.0	35.3
III/300	22.3	12.4^#	0.1	9.3	17/18	94.4	6.2	9.3	26.0	36.4	83/75	1.11
IV/1000	22.1^#	11.1	0.0	8.8	19/19	100	5.8	9.2	24.8	34.7	84/84	1.00
V/3000	22.2	11.7	0.8	9.5	13/13	100	5.8^#	9.2	25.6	34.6	60/63	0.95
F1 to F2b
I/0	22.3	12.3	0.1	8.9	14/15	93.3	6.0	8.8	26.1	39.2^+	59/66	0.89
II/0	22.1	9.4	0.9	7.8	15/17	88.2	6.1	9.7	29.5	45.0*	61/56	1.09
I+II	22.2	10.8	0.6	8.3			6.0	9.3	27.8
III/300	22.1	12.5	0.3	9.3	19/19	100	6.1	9.5	27.6	41.3	83/93	0.89
IV/1000	22.1	11.4	0.9	9.3	15/17	88.2	5.8	9.0	26.5	39.5 ^++	75/73	1.03
V/3000	22.2	11.1	0.5	9.2	14/15	93.3	5.9	9.3	27.2	40.1	63/66	0.95

Significantly different from control Group I: *p≤0.05; **p≤0.01.

Significantly different from control Group II: +p ≤0.05; ++p ≤0.01.

Significantly different from pooled values of control Groups I and II: #p ≤0.05; ##p ≤0.01.

a Pre-cull weight only.

b Data not analysed statistically.

Conclusions:

In a well conducted and documented, pre-GLP one generation reproductive toxicity study (reliability score 2), CP 66257 (DTPMP) administered via the diet caused no clear treatment-related or statistically significant effects in rats. Pregnancy rate and pup body weight were lower for F2a litters from dams fed diets containing 3000 ppm DTPMP, but this was not replicated in the F1 or F2b litters. A NOAEL of 294 mg/kg bw/d for males and 312 mg/kg bw/d in females was therefore concluded.

Executive summary:

In a well conducted and documented, pre-GLP one generation reproductive toxicity study (reliability score 2), CP 66257 (DTPMP) was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. Parameters evaluated included parental mortality, mating-fertility indices, body weight and food consumption data, necropsy observations and organ weight data (absolute and relative) for the F1 parental generation. Litter parameters included gestation length, pup viability at birth and litter survival indices. Pups were evaluated for survival, growth and necropsy observations at weaning.

In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant). Pups also had a lower weight at birth (

not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. In the mid dose group, pup weight at birth was lower than control in the first litters (F2a) only. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 294 mg/kg bw/d for males and 312 mg/kg bw/d in females was therefore concluded.

Reference 5

Endpoint:

two-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

29.10.1976 to 15.08.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Three generation reproduction toxicity study with the following restrictions: no assessment of estrus cycle, sperm parameters, sexual milestones, no analytical confirmation of exposure levels.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, New York.
- Age at study initiation: (P) 6-7 wks
- Weight at study initiation: (P) males approx. 370 g, females approx. 240 g at mating
- Fasting period before study: No data
- Housing: Individually (except during mating and lactation) in elevated stainless steel wire mesh cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data


IN-LIFE DATES: From: 12.11.1976 To: 15.08.1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow (standard laboratory diet)
- Storage temperature of food: No data

Details on mating procedure:

- M/F ratio per cage: 1/2 (See table 1)
- Length of cohabitation: Overnight for up to 15 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Remated (for F1b/F2b/F3b generation) following a 14 d rest period.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually in elevated stainless steel wire mesh cages.
- Any other deviations from standard protocol: None apparent.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet samples were taken from control and treated groups weekly. Samples were stored frozen and sent to the sponsor at regular intervals throughout the study. No further details.

Duration of treatment / exposure:

From 60 days prior to first mating of P generation then continuous over 3 generations . Duration of test in total was approximately 21 months.

Frequency of treatment:

Daily

Details on study schedule:

- F1 and F2 parental animals not mated until after a growth period (unspecified duration) following selection from the F1b and F2b litters.
- Selection of parents from F1 and F2 generation when pups were 7 days post weaning.
- Age at mating of the mated animals in the study: Not clear, but there was a 14 day rest period between matings.

Dose / conc.:

300 ppm

Remarks:

nominal in diet
See table 3 for conversion to mg/kg bw/day

Dose / conc.:

1 000 ppm

Remarks:

nominal in diet
See table 3 for conversion to mg/kg bw/day

Dose / conc.:

3 000 ppm

Remarks:

nominal in diet
See table 3 for conversion to mg/kg bw/day

No. of animals per sex per dose:

12 male and 24 females (see Table 1)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random

Positive control:

None

Parental animals: Observations and examinations:

Examination conducted on F0, F1, F2 AND F3 (all adult generations)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Gross signs twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during growth and rest periods of all animals. As well as pregnant females (F0, F1b, F2b) on GD 0, 6, 15 and 20 and lactating females (F0, F1) on LD 0, 4, 14 and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly for males and non-pregnant females.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: F0 parents after weaning of F1b litter.

Oestrous cyclicity (parental animals):

Not investigated.

Sperm parameters (parental animals):

Not investigated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, to 10 pups/sex/litter as nearly as possible; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities. See Tables 4, 5 and 6 for pup survival data.


GROSS EXAMINATION OF DEAD PUPS:
yes, sex determined and stomach checked for presence of milk. Cause of death was not determined.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation.
- Maternal animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation. Also non-pregnant dams from first mating.
- Dead and moribund animals examined as death occurred.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dams uterine contents examined for the presence of implantation sites and/or scars.


HISTOPATHOLOGY / ORGAN WEIGHTS: None scheduled for adults.Only grossly abnormal tissues were examined.

Postmortem examinations (offspring):

SACRIFICE
- The F1a/F2a/F3a offspring not selected as parental animals were sacrificed at 21 days of age.
- F1b and F2b progeny (non-parental): sacrificed after pup selection for next generation.
- F3b sacrificed at weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination from 10 pups/sex/group of the F3b generation. In addition any grossly abnormal tissues were examined.

Statistics:

Offspring body weights, off-spring numbers (LD 0 LD 4): F-test and Student's T-test.
Offspring survival, litter deaths, litters weaned, mortality, mating rates, pregnancy rates, fertility rates: Chi square.
Body weights, body weight change, food intake: Dunnett's test.

Reproductive indices:

mating indices (%), pregnancy rates (%) and fertility (%). No details given.

Offspring viability indices:

No details given.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): One mid dose male was sacrificed in a moribund state during mating for F1b litter ("severely tilted head"). One high dose female was sacrificed post-weaning of F1b litter with "extremely distended abdomen".  F1: One mid dose male died during mating. One high dose female died during post-weaning interval, prior to sacrifice. One control male and one high dose female sacrificed with "severely tilted heads" (male sacrificed wk 4, female in rest period between matings). F2: One low dose female died during wk 3 of the growth period. One mid dose male died during wk 8 of the growth period. One mid dose female died on GD 21 for the F3b litter. One high dose male died during the mating interval to produce the second litter. The was no dose response and the deaths were considered sporadic, therefore not related to treatement.
Physical observations comparable between all groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There was no effect in either sex on mean body weight or body weight gain during growth or rest periods. There were no effects on maternal body weight or body weight change during gestation or lactation periods.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): See Table 3


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Gestation length, mean number of live and dead pups at birth and percentage of live pups at birth were comparable between groups. A high number of dead pups within a single mid dose litter lead to a significant decrease in the survival index at birth in the mid dose group for F1b. The F2 generation high dose group had a significantly higher survival index at birth for the second litters. No adverse effect was concluded. There was no effect on mating indices (%), pregnancy rates (%) or fertility (%).


GROSS PATHOLOGY (PARENTAL ANIMALS): There were no adverse findings.


HISTOPATHOLOGY (PARENTAL ANIMALS): Not conducted.


OTHER FINDINGS (PARENTAL ANIMALS): The ophthalmoscopic examination revealed four rats (one control female, two mid dose males and one high dose male) with ocular abnormalities. However, these were not considered to be treatment related.

Dose descriptor:

NOAEL

Effect level:

>= 275 mg/kg bw/day

Sex:

male

Basis for effect level:

other: corresp. to >= 3000 ppm

Dose descriptor:

NOAEL

Effect level:

>= 310 mg/kg bw/day

Sex:

female

Basis for effect level:

other: corresp. to >= 3000 ppm

Dose descriptor:

other: NOAEL F3

Effect level:

>= 275 mg/kg bw/day

Sex:

male

Basis for effect level:

other: corresp. to >= 3000 ppm

Remarks on result:

other: Generation not specified

Dose descriptor:

other: NOAEL F3

Effect level:

>= 310 mg/kg bw/day

Sex:

female

Basis for effect level:

other: corresp. to >= 3000 ppm

Remarks on result:

other: Generation not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING): Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups, however these were not considered adverse by the authors since no trend was present.  Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.

CLINICAL SIGNS (OFFSPRING): None reported.


BODY WEIGHT (OFFSPRING): Comparable between all groups.


GROSS PATHOLOGY (OFFSPRING): No adverse findings.


HISTOPATHOLOGY (OFFSPRING): scattered red foci present in lung from some F2b offspring from the mid and high dose groups (not present in controls and low dose group) considered unrelated to treatment by authors (since not present in other generations). All other necropsy observations similar for control and treated litters. Evaluation of selected tissues from 10 control weanlings and 10 high dose weanlings from the F3b generation revealed no abnormalities. Changes present in lung consistent with minimal to mild interstital pneumonia, microscopic appearance of the gonads unremarkable and consistent with sexually immature rats.

OTHER LITTER PARAMETERS: Sex ratio was not affected by treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 275 mg/kg bw/day

Sex:

male

Basis for effect level:

other: corresp. to >= 3000 ppm

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 310 mg/kg bw/day

Sex:

female

Basis for effect level:

other: corresp. to >= 3000 ppm

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 275 mg/kg bw/day

Sex:

male

Basis for effect level:

other: corresp. to >= 3000 ppm

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 310 mg/kg bw/day

Sex:

female

Basis for effect level:

other: corresp. to >= 3000 ppm

Reproductive effects observed:

not specified

Table 3 Test substance intake based on food intake (weekly mean data) measurements .

	Males (mg/kg bw/day)			Females  (mg/kg bw/day)
Group (ppm)	II (300)	III (1000)	IV (3000)	II (300)	III (1000)	IV (3000)
F0 growth	33.4	111.6	342.3	37.3	117.1	362.5
F0 rest	18.4	60.7	182.9	24.3	75.7	247.2
F1 growth	26.3	89.6	270.2	28.1	98.4	290.2
F1 rest	14.4	41.7	141.0	20.1	67.3	201.1
F2 growth	29.6	95.4	296.6	34.3	112.6	337.8
F2 rest	17.6	58.3	171.9	25.0	81.4	243.9

Table 4 Summary of offspring survival for the F1 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)			% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F0 to F1a
I (0)	22.5	98.8	9.1	92.0	94.3	100	57.1	95.2	0.87
II (300)	22.4	98.2	8.6	97.3*	90.5	100	65	100	0.87
III (1000)	22.5	98.1	7.6*	96.2	85.4**	100	47.6	95.2	0.88
IV (3000)	22.3	98.1	8.7	99.2**	91.4	99	52.2	100	1.24
F0 to F1b
I (0)	22.1	93.0	8.9	87.2	96.0	99.3	61.1	88.9	1.07
II (300)	22.1	96.0	8.7	91.7	91.3	99.4	83.3	100	1.12
III (1000)	22.1	97.2	9.3	94.8*	93.7	100	56.3	100	0.84
IV (3000)	22.1	95.8	9.2	97.6**	96.5	99.5	28.6	100	0.96

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 5 Summary of offspring survival for the F2 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)			% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F1 to F2a
I (0)	22.2	99.5	9.2	96.2	93.5	100	33.3	94.4	1.09
II (300)	22.3	100	9.5	96.8	99.5**	100	35.0	100	1.18
III (1000)	22.1	99.6	9.4	95.4	99.5**	98.6	40.9	100	1.06
IV (3000)	22.3	97.4	9.1	97.3	100**	100	30.4	100	0.92
F1 to F2b
I (0)	22.4	99.3	8.8	78.5	100	100	35.7	85.7	0.89
II (300)	22.1	96.1	8.8	93.6**	92.5	98.1	61.1	100	1.11
III (1000)	22.1	92.5**	8.2	92.4**	88.5**	58.8	93.8	93.8	0.95
IV (3000)	22.5	99.1	9.0	96.6**	97.8	98.9	50.0	100	0.94

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 6 Summary of offspring survival for the F3 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)			% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F2 to F3a
I (0)	22.3	97.9	7.8	89.5	94.3	99.4	54.5	95.5	0.91
II (300)	22.4	91.3	6.4	87.2	89.5	100	75.0	100	1.13
III (1000)	22.2	96.2	7.7	85.9	89	100	57.1	87.7	1.09
IV (3000)	22.2	98.6	8.8	96.7**	91.9	99.4	66.7	94.7	1.11
F2 to F3b
I (0)	22.2	92.9	9.1	89.5	97.4	98.6	50.0	88.9	1.00
II (300)	22.4	96.1	8.7	92.4	98.5	99.2	43.8	93.8	1.06
III (1000)	22.1	92.7	8.3	93.5	97.3	100	38.5	100	0.83
IV (3000)	22.2	98.5**	9.2	89.9	98.7	100	52.9	94.1	1.45

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Conclusions:

In a reasonably well conducted three generation reproductive toxicity study conducted before the adoption of OECD test guidelines and GLP, the general and reproductive toxicity NOAEL for ATMP was greater than the highest dose tested, 3000 ppm in the diet, in rats (approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day for females).

Executive summary:

Male and female Long-Evans rats were administered CP 42902 (nitrilotrimethylenetris(phosphonic acid)) continuously in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The litters from F0, F1 and F2 matings were raised to maturity and also mated. Offspring from the first litter of each generation (F1a, F2a, F3a) were taken for necropsy on lactation Day 21. The parents were remated following a 14 d rest period and the offspring randomly selected at seven days post-weaning to continue as the F1b and F2b generation parents. Remaining F1b and F2b animals, as well as the F3a and F3b generation, were taken for necropsy. A gross internal axamination was conducted on these animals. Randomly selected offspring from the F3a litters (10 pups/sex/group) were necropsied and selected tissues examined microscopically. Evaluations of adult mortality, mating, pregnancy, fertility, body weight data, food consumption data (growth and rest periods), litter survival, offspring viability at parturition, offspring weight and sex, and necropsy of adults and offspring, did not indicate any treatment-related adverse effects. The NOAEL for general toxicity and reproductive toxicity was greater than the highest dose tested, 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. 3000 ppm was approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day in females.

Additional information

The substance under registration (CAS 3794-83-0) can chemically be described as a tetrasodium salt of HEDP. Information on reproduction toxicity is not available for this specific sodium salt, but data are available for other sodium salts of HEDP which belong to the same category. Altogether, six studies covering several study types (90-day study, combined chronic tox./carcinogencity study, dominant-lethal-assay, two-generation reproduction study) are available which provide information on fertility. The tested species were rats, dogs and mice. In the two-generation key study, oestrus cycles but not sperm parameters were assessed. However, no effects on spermatogenesis or testes were observed in the second key study (combined chronic toxicity/carcinogenicity test) or subchronic studies.

In all studies, no effects on reproductive functions were observed up to the highest dose level tested. An overview on all studies and results is given in the table below.

Entry	Test type / species/ author	Klim.	Scope (reproduction parameters)	Deficiencies	Result (Fertility)
1 Key	Two-generation reproduction toxicity study (rat)   Nolen & Buehler, 1971	2	Pregnancy rate, number of resorptions, corpora lutea and implantations, number of live/dead fetuses, weight of fetuses/pups/dams; determination of estrus cycles by vaginal smear, feeding from week 5 onwards	Use of 2 instead of three dose levels; Sexual milestones in pups/analytical and confirmation of dose not given; No sperm analyses. Not GLP.	NOAEL = 112 mg/kg bw/d
2 Key	Combined chronic toxicity / carcinogenicity study (rat)   Huntingdon Research, 1979	2	Organ weights and histology of testes, seminal vesicles, prostate, uterus, mammary (gland); Spermatogenesis, atrophy, dilation of tubules, periarteritis	Only 40 instead of 50 animals were used. Not GLP.	NOAEL >= 384 mg/kg/day in male rats and >= 493 mg/kg/day in female rats.
3 Supp	90-day study oral toxicity study (rat),   Huntingdon Research, 1977	2	No observable effect, with respect to the testes, ovaries and associated organs	Comparable to OECD, not GLP	NOAEL >= 817 mg/kg bw/d (m) or >= 1000 mg/kg bw/d (f)
4 Supp	Dom.- lethal- test (mouse),   Pieper K., 1974	2	Conception rates, total implant averages, fetal death averages, resorption percentage, mutagenic indices	Postive control substance was not included in this study (not necessarily required). Not GLP.	Not toxic to reproduction in doses up to 1000 mg/kg bw/d, (given 5 times, on 5 consecutive days)
5 Supp	90-day oral toxicity study (dog),   Industrial Biotest, 1975	2	Organ weights of gonads, histopathology of gonads (males: testes and epididymides), prostate gland and uterus	Pre-GLP/-OECD.	NOAEL 1746 mg active acid/kg bw/d (males) and 1620 mg active acid/kg bw/d (females)
6 Supp	90-day oral toxicity study (rat)   Marias, A., 1975	2	Organ weights of gonads, microscopic examination of tissues (gonads, seminal vesicles, uterus, prostate gland)	Not GLP	NOAEL >= 900 mg/kg bw/d

 

Further information on fertility is available from reproduction toxicity studies conducted with the structurally homologue phosphonates DTPMP-H (CAS 15827-60-8, entry 7), ATMP-H (CAS 6419-19-8, entry 8) and HEBMP-xNa (CAS 22036-78-8, entry 9). Those studies support in a weight-of-evidence approach the hypothesis that phosphonates do not induce specific effects on fertility. An overview on these data obtained with structurally related phosphonates is given in the table below.

 

Entry	Test type / species / CAS	Klim.	Scope (reproduction parameters)	Deficiencies	Result (Fertility)
7 WOE	One generation study (rat)	2	Organ weights and histology of testes, seminal vesicles, prostate, uterus, mammary (gland); Spermatogenesis, atrophy, dilation of tubules, periarteritis	No analytical confirmation of exposure, no pre-mating exposure, no assessment of oestrus cycle or sperm analyses, no evaluation of sexual milestones in pup. Males treated only as F1 and F2 generation.	NOAEL (P) >= 882 mg/kg bw/d (m) and >= 936 mg/kg bw/d (f) NOAEL (F1, F2) = 294 mg/kg bw/d (m) and 312 mg/kg bw/d (f)
8 WOE	Three-generation reproduction study (rat)	2	Organ weights and histopathology of gonads	No assessment of estrus cycle, sperm parameters, sexual milestones, no analytical confirmation of exposure levels. Not GLP.	NOAEL >= 275 mg/kg bw/d (m) and >= 310 mg/kg bw/d (f)
9 WOE	OECD 422 (rat)	1	Conception rates, total implant averages, fetal death averages, resorption percentage, mutagenic indices	None	NOEL >= 1000 mg/kg bw/d

 

Justification for grouping:

See CSR Annex I and II or IUCLID section 13.

Effects on developmental toxicity

Description of key information

Regarding sodium salts of HEDP a combined two-generation study of reproductive toxicity and teratogenicity in rats as well as a prenatal developmental toxicity study in rabbits was published in the peer reviewed literature (Nolen & Buehler, 1971).  Several related phosphonic acid compounds from the ATMP, HEBMP, and DTPMP categories are not teratogenic following oral treatments in rats and mice between 312 and >= 1000 mg/kg bw/day during pregnancy. In conclusion, all available data have not revealed any adverse effects on development in any of the tested species (rat, rabbit, mouse). On the basis of the key study with the most sensitive species (rabbit), and in consideration of the overall weight of evidence, a developmental NOAEL of >= 100 mg/kg bw/day appears appropriate for HEDP and its salts.

Link to relevant study records

Reference

Reference 1

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

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

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.

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

Dose / conc.:

0.1 other: %

Remarks:

nominal in diet

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.

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).

Dose descriptor:

NOAEL

Effect level:

112 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Dose descriptor:

NOAEL

Effect level:

447 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

12.09.1977 to 19.06.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 2c The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Females: Blue Spruce Farms. Males: BioDynamics Inc. in-house Long Evans breeding colony.
- Age at study initiation: (P) x 15-16 wks; (F1) x 3 wks
- Weight at study initiation (means): Females approximately 240 g. Males not weighed.
- Fasting period before study: No
- Housing: Individually in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12.09. 1977 to 19.06.1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow
- Storage temperature of food: No data

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: nightly until signs of mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually in elevated stainless steel cages.
- Any other deviations from standard protocol: Dosing not started until Day 0 of gestation.

Duration of treatment / exposure:

Test substance administration to F0 females was initiated on gestation Day 0 and continued throughout gestation and lactation, and for F1males and females through the F2a and f2b litters.

Frequency of treatment:

daily

Duration of test:

281 days

Dose / conc.:

300 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

3 000 ppm (nominal)

No. of animals per sex per dose:

F0 females: 20.
F1 Parents: females - 20; males - 10.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random.
- F1 offspring were separated from siblings seven days after weaning of the last litter and were randomly selected to continue as future parents (f1). More offspring than needed were selected (12 males and 24 females) for the growth period to insure the required number of adults (10 males and 20 females) necessary for mating. Following pup selection, remaining offspring and F0 females were sacrificed and discarded after gross external and internal examinations.
F1 animals were raised to maturity and mated to produce the F2a litters. F2a pups were sacrificed, necropsied and discarded at weaning. All F1 females were remated after a rest period of at least 14 days to produce the F2b litters. F2b pups were sacrificed and necropsied at weaning. Following completion of the F2b sacrifice, all F1 parents were sacrificed, necropsied and selected tissues preserved.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and gross signs of toxicity in F0 and F1: twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly (F0 and F1)


BODY WEIGHT: Yes
- Time schedule for examinations: Nonpregnant females (F1): weekly in growth and rest periods of F1 generation. Pregnant females (F0 and F1): Days 0, 6, 15 and 20 of gestation. Lactation females (F0 and F1): Days 0, 4, 14 and 21 of lactation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Nonpregnant females: weekly for growth and rest periods of F1 generation.
-Test substance consumption was calculated from body weight and food consumption values: Nonpregnant females: weekly during growth and rest periods of F1 generation.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Ovaries and uterine content:

Not applicable to this study - See Section 7.8.1 for examinations conducted.

Fetal examinations:

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning. Necropsy performed including internal sex determination peformed, then carcassed discarded.
- F2a: necrospy included internal sex determination performed. All offspring where then discarded.
- F2b: necrospy included internal sex determination performed. Selected tissues were preserved from ten randomly chosen males and females from all groups.
F2b found dead after Day 14 of lactation: necropsy including internal sex determination performed and discarded.
Culled F1, F2a and F2b: sacrifice on Day 4 of lactation. Necropsy including internal sex determination performed and discarded.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dead pups: sex was determined and pups checked for presence of milk in their stomach and discarded.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 in Section 7.8.1 were preserved for F2b offspring.

Statistics:

Body weight, body weight gain, food consumption and litter examination data, organ weights, organ/body weight and organ/brain weight ratios were analysed. Group I and Group II (control groups) were compared to each other. If no statistically significant differences, the control groups were combined and compared against test groups. If controls differed, individual control/test comparisons conducted - continuous and discrete data (body weight, food intake, organ weight): Dunnett's test.  - gestation length: F-test and Student's T-test (Cochran's approximation). - offspring data: Chi-square. Incidence data for control Group II was compared to control Group I and incidence data for each test substance treated group was compared to each control group.

Indices:

See Section 7.8.1

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

312 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no teratogenic effects observed in offspring.
Reduced birth weight was evident in some offspring but this was not replicated in all generations.

Dose descriptor:

NOAEL

Effect level:

312 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a well conducted and documented, pre-GLP one generation reproductive toxicity study (reliability score 2), CP 66257 (DTPMP) administered via the diet caused no clear treatment-related or statistically significant effects in rats. A NOAEL of 312 mg/kg bw/d for maternal toxicity and developmental toxicity was therefore concluded.

Executive summary:

See Section 7.8.1 for study Executive Summary.

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

No data. Dates of treatment were 27.12.1978 to 19.01.1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Guideline:

other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)

Deviations:

not specified

Remarks:

Treatment on GD 6 - 15; no record of gravid uterine weight; number corpora lutea not recorded; no analytical confirmation of exposure levels.

Principles of method if other than guideline:

Study was used to assess the teratogenic and/or embryotoxic potential of the test substance.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Further matings after two unsuccessful attempts: No data
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 - 15

Frequency of treatment:

daily

Duration of test:

16 days

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 21 d

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

MATERNAL TOXIC EFFECTS BY DOSE LEVEL AND BY SEX
Effects with dose level: 0/100/500/1000 mg/kg bw/d

Mortality
- one female from 100 mg/kg bw/d group moribund and sacrificed on GD6 (first day of treatment)

Body weight
- no significant differences in maternal bwt gain between groups
- body weight gain GD6-15: 50/49/50/44
- 12% (non-significant) reduction in body weight gain at 1000 mg/kg bw/d on GD6-15
Comment: individual body weight gain for dam 822 (high dose) on GD6-15 = 22 g; mean gain for controls = 50 g; mean gain for high dose group = 44 g. Bwt gain for this dam on GD 0-6 (preceding treatment) and on GD 15-21 (post-treatment) was similar or greater than mean bwt gain for control and high dose group.

Clinical observations, physical signs
- none present

Necropsy findings
- few adverse changes present, no treatment related effects 

Reproductive parameters
- pregnancy rate comparable between groups (100% in control, mid and high dose groups, 95.6% at 100 mg/kg bw/d), no
treatment-related changes.
- mean number of corpora lutea (17.2/16.0/16.7/16.8), implantations (14.5/14.7/14.0/13.7) and implantation efficiency (84.3%/91.8%/84.0%/81.2%) comparable; significant 7% decrease in corpora lutea and significant 7.5% increase
in implantation efficiency at 100 mg/kg bwt/d (P<0.05 in both instances) considered unrelated to treatment by authors
- mean number live fetuses comparable: 14.3/13.9/13.5/12.9 (no dead fetuses in any group)
- mean number resorptions: 0.2/0.8(P<0.05)/0.5/0.8(P<0.05); all values within historical control range
- non-dose related, non-significant increase in dams with 2 or more resorptions in treated groups (0%/22.7%/8.3%/20.8%);
within historical control range

FETAL DATA
Effects with dose level: 0/100/500/1000 mg/kg bw/d
- body weight by sex: males 5.54/5.43/5.71/5.49; females 5.25/5.17/5.34/5.16 (no significant effect)

Crown-rump length: males 4.2/4.2/4.3 (P<0.01)/4.2; females 4.1/4.1/4.2 (P<0.01)/4.1 (increase at 500 mg/kg bw/d
considered biologically insignificant by authors)

Sex: males/litter 6.9/7.0/6.6/6.0; females 7.4/6.9/7.0/6.9 (no significant effect)

Sex ratio (m:f): 92.4%/102.0%/94.6%/87.3% (no significant effect)

Variation in ossification: fetuses 80.2%/83.5%/79.3%/84.3%; litters 95.8%/100.0%/100.0%/100.0% (no significant effect)

External malformations: 
- incidence: 0 fetuses from 339 examined/0 from 305 examined/0 from 325 examined/6 from 315 examined
-  in the high dose group, one female (no. 822) had 6 fetuses (from a total litter of 16) with a syndrome of
defects that included: flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated
forward flexure of the head (see maternal body weight, above). Remaining high dose fetuses (n = 309) unremarkable.

Total skeletal malformations 
- total fetuses examined: 177/158/169/159
- per fetus: 4.0%/1.9%/3.0%/1.3% (no significant effect)
- per litter: 20.8%/13.6%/20.8%/8.7% (no significant effect)
- type of skeletal malformations control: angulated ribs, cervical rib, wavy rib
100 mg/kg: angulated rib, cervical rib, angulated and wavy rib
500 mg/kg: cervical rib, angulated and wavy rib, 7 lumbar vertebra
1000 mg/kg: 5 lumbar vertebra, fused sternebrae 

Total soft tissue malformations
- total fetuses examined: 162/147/156/150
- per fetus: 4.3%/8.2%/4.5%/4.0% (no significant effect)
- per litter: 16.7%/40.9%/29.2%/20.8% (no significant effect)
- type of soft tissue malformation
control: distended renal pelvis, renal pelvis, ureter, baldder
100 mg/kg: as control + fold in retina
500 mg/kg: as control + ectopic kidney
1000 mg/kg: as control + fold in retina, anophthalmia, malrotation of heart
- malrotation of the heart occurred in high dose 2 fetuses, both from litter No. 822 (see "maternal body weight"  and
"external malformations", above)

Visceral malformations
- per fetus: 0.6%/1.3%/0.6%/1.9% (no significant effect)
- per litter: 4.2%/9.1%/4.2%/12.% (no significant effect)
- type of visceral malformation control: distended ureter
100 mg/kg: distended ureter +/- renal pelvis 500 mg/kg: as control
1000 mg/kg: as control + malpositioned testis

Conclusions:

In a well documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) Dequest 2000 was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg/kg bw/d by gavage on GD6-15. At 1000 mg/kg bw/d, six fetuses from a single litter showed common multiple malformations in presence of a 50% decrease in individual maternal body weight gain (possibly indicative of concurrent maternal toxicity); all other high dose fetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response indicates that 1000 mg/kg bwt/d was a probable no-effect level for embryotoxicity and fetotoxicity. The maternal NOAEL was 500 mg/kg bw/day.

Executive summary:

In a well documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) Dequest 2000 was administered by oral gavage to pregnant Charles River CD rats (24/dose), at dose levels of 100, 500 and 1000 mg/kg bw/day, on gestation days 6 to15. Control animals received the vehicle (water) only. Dams were sacrificed on gestation day 21 and recovered fetuses evaluated for external, soft-tissue and skeletal malformations. Maternal mortality, pregnancy rate, body weight gain, uterine implantation data, fetal size, sex data, ossification variation data and teratological evaluations were evaluated. High dose females gained less weight than the controls during the dosing period. A statistically significant increase in the number of resorptions was observed in the low and high dose animals (not the mid-dose). There was also an increase in the number of dams with two or more resorptions. However, the resorption data were within the range of historical values for the laboratory, so it was concluded that there was not a treatment-related effect. There were no teratogenic effects in the low and mid dose group. In the high dose group six fetuses from a single litter had common multiple malformations that included flexed forepaws, shortened and thickened torso, abdominal distention and exaggerated flexure of the head. Soft tissue examination revealed two of these fetuses had a malformation defect of the heart. The remaining high dose fetuses were generally unremarkable. Soft tissue and skeletal malformation data from the high dose group were similar to the control group. Although a possible teratogenic effect could not be excluded, it was most likely that the effects were secondary to maternal toxicity. Therefore the maternal NOAEL was 500 mg/kg bw/day, and the NOAEL for fetotoxicity and teratogenicity was >1000 mg/kg bw/day.

Reference 4

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

No data. Treatment days were 09.04.1980 to 13.05.1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Guideline:

other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)

Deviations:

not specified

Remarks:

No analytical evaluation of exposure levels.

Principles of method if other than guideline:

The study was designed to evaluate the embryotoxic and/or teratogenic potential of the test substance. Dosing was on gestation days 6-15, no measurement of gravid uterine weights and copora lutea were not counted.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study initiation: Females 57 days (males stated to be sexually mature)
- Weight at study initiation: Females on gestation Day 0 were approximately 26 g.
- Fasting period before study: No data
- Housing: Individual in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: One month


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data. Monitored twice daily.
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: Day 6 of gestation: 09.04.1980 - 04.05.1980 To: Day 15 of gestation: 18.04.1980 - 13.05.1980.

Route of administration:

oral: gavage

Vehicle:

other: Not clear, stated to be distilled water and corn oil in different parts of the report.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance were dissolved in distilled water and administered at a constant volume of 10 ml/kg bw/day. Dosing solutions were prepared fresh daily.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 - 15

Frequency of treatment:

daily

Duration of test:

18 days

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

35 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes for mortality and gross signs of toxicological effects (no further details).
- Time schedule: Twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation days 0, 6, 9, 12, 15 and 18.


BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 9, 12, 15 and 18. Calculated body weight change for days 0-6, 6-15 and 15-18.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 (all surviving dams) and Day 18 post-mating in all surviving non-pregnant females.
- Organs examined: Complete gross pathology examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes, and weighed, measured and sex determined.
- Other: Live and dead fetuses. Internal sex determination of fetuses.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No data

Statistics:

Maternal body weight and reproduction data: Bartlett's test followed by one-way ANOVA (equal variance) followed by Dunnett's test or Kruskal-Wallis test (unequal variance) and summed rank test (Dunn).  Pregnancy and fetal parameters: Chi square analysis followed by Fisher Exact test with Bonferroni correction. Armitage test for linear trend.

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on mean fetal body w eights, crown-rump length and fetal sex distribution. During the fetal skeletal evaluations, the incidence of fetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However there was a slight increase in the incidence of fetuses with rudimentary structures observed in the mid and high dose groups. The incidence of fetal external and soft tissue malformations were comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed fetuses from a single mid-dose litter. Six fetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 Summary of Malformations found during the fetal skeletal examination.

Group (mg/kg bw/day)	Malformation	Fetuses		Litter
		No. with malf./total examined	%	No. with malf fetuses/total examined	%
0	None	0/138		0/24
100	Cervical vertebral defect	1/156	0.6	1/28	3.6
500	Tibia misshapened - alone	1/203	0.5	1/34	2.9
	- with misshapened fibula and angulated ribs	2/203	1.0	1/34	2.9
	- with misshapened fibula, angulated ribs and sternebrae defects	1/203	0.5	1/34	2.9
	- with angulated ribs	1/203	0.5	1/34	2.9
	Angulated ribs and scrambled sternebrae	1/203	0.5	1/34	2.9
	Cervical rib	1/203	0.5	1/34	2.9
	5 lumbar vertebrae	2/203	1.0	1/34	2.9
	Total	9/203	4.4*	3/34	8.8
1000	Scrambled sternebrae	1/183	0.5	1/32	3.1
	Vertebral defects	1/183	0.5	1/32	3.1

*Difference from the control group statistically significant p<0.05 (Fisher Exact test).

Conclusions:

In a well conducted teratogenicity study (FDA segment II: teratological study; reliability score 2) conducted prior to the adoption of OECD test guidelines and GLP, it was concluded that Dequest 2000 was not embryotoxic or teratogenic when administered to mice at 100, 500 or 1000 mg/kg bw/d
by gavage on GD6-15.

Executive summary:

In a well conducted teratogenicity study (FDA segment II: teratological study; reliability score 2) conducted prior to the adoption of OECD test guidelines and GLP, Dequest 2000 was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6 -15. The doses tested were 100, 500 and 1000 mg/kg bw/day. The control group received the vehicle only. Parameters evaluated were mortality, body weight, clinical signs, uterine implantation data, ossification variation data and teratological evaluation. There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination. No effects on mean fetal body weights, crown-rump length and fetal sex distribution. During the fetal skeletal evaluations, the incidence of fetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However there was a slight increase in the incidence of fetuses with rudimentary structures observed in the mid and high dose groups. The incidence of fetal external and soft tissue malformations were comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed fetuses from a single mid-dose litter. Six fetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment. The NOAEL for maternal toxicity, fetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

25.10.1979 to 17.12.1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Study conducted prior to the adoption of OECD test guidelines.

Deviations:

yes

Remarks:

Study is only a pilot so examinations are limited.

Principles of method if other than guideline:

This pilot study was designed to establish doses for an intended Segment II teratology study.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 15 of pregnancy.

Frequency of treatment:

Daily.

Duration of test:

13 days

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Ten

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 Reproduction data from gestation Day 18.

Group (mg/kg bw/day)	No. pregnant females	Mean No. implantations	Mean No. fetuses			Mean No. resorptions	Dams with 2 or more resorptions
			live	dead			total	%
I (0)	5^a	10.4	9.6	0.2		0.6	1/5	20.0
II (100)	7^a	10.1	9.3	0		0.9	1/7	14.3
III (500)	4	10.5	9.5	0		1.0	1/4	25.0
IV (1000)	8	10.5	9.8	0		0.8	2/8	25.0

a Excludes one pregnant female that delivered a litter prior to sacrifice.

Conclusions:

In a pre-GLP, pilot teratology study (reliability score 2) conducted prior to the adoption of OECD test guidelines, no adverse effects on dams or fetuses were observed. The NOAELs for maternal toxicity and teratogenicity were at least 1000 mg/kg bw/day in mice.

Executive summary:

In a pilot teratology study, FA42902 (Dequest 2000) was administered by oral gavage to pregnant CD-1 mice from Day 6 to 15 of gestation at doses of 100, 300 or 1000 mg/kg bw/day. Control animals received equal volumes of distilled water. Females were sacrificed on gestation Day 18 and fetuses were examined for external malformations. Maternal mortality, body weight gains, implantation data, in-life physical observations and gross postmortem examination data, fetal body weights, sex distribution and external malformation data did not reveal any signs of an adverse toxicological effect. No obvious or consistent test substance-related effects on reproduction were observed at any dose. The NOAELs for maternal toxicity and teratogenicity were at least 1000 mg/kg bw/day.

Reference 6

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

08.06.1978 to 22.06.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Study conducted prior to the adoption of OECD test guidelines.

Deviations:

yes

Remarks:

Study is a pilot so examinations are limited.

Principles of method if other than guideline:

This pilot study was designed to establish doses for an intended Segment II teratology study.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: Overnight
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 15 of gestation

Frequency of treatment:

Daily

Duration of test:

16 Days (Animals sacrificed on gestation day 21)

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 Reproduction data on gestation Day 21.

Group (mg/kg bw/day)	Mean No. corpora lutea	Mean No. implantations	Implantation efficiency^a		Mean No. live fetuses	Mean No. resorptions	Dams with 2 or more resorptions
			total	%			total	%
I (0)	13.4	12.4	62/67	92.5	12.0	0.4	0/5	0
II (100)	13.6	12.4	62/68	91.2	12.2	0.2	0/5	0
III (500)	12.8	11.5	46/51	90.2	10.8	0.8	0/5	0
IV (1000)	13.6	11.2	56/68	82.4	10.4	0.8	2/5	40

a Implanation efficiency was calculated by dividing the total number of implantation sites by the total number of corpora lutea and multiplying the result by 100.

Conclusions:

In a pre-GLP, pilot teratology study (reliability score 2) conducted prior to the adoption of OECD test guidelines, no adverse effects on dams or fetuses were observed. The NOAELs for maternal toxicity and teratogenicity were at least 1000 mg/kg bw/day in rats.

Executive summary:

In a pilot teratology study, FA42902 (Dequest 2000) was administered by oral gavage to pregnant Long-Evans rats from Day 6 to 15 of gestation at doses of 100, 500 or 1000 mg/kg bw/day. Control animals received equal volumes of distilled water. Females were sacrificed on gestation Day 21 and fetuses were examined for gross findings. One dam from the mid dose group was found dead on Day 13 of gestation. Mean body weight gain was slightly lower than controls for the mid and high dose groups during the treatment period, and lower than controls on gestation Days 15 to 21. No obvious or consistent test substance-related effects on reproduction were observed at any dose. Maternal physical pbservations and necropsy data, and fetal gross findings did not reveal evidence of adverse effects. The NOAELs for maternal toxicity and teratogenicity were at least 1000 mg/kg bw/day.

Reference 7

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Publication available for review, non-guideline study, poorly described methods and results, insufficient for full assessment, non-physiological route of exposure, low number of animals (6-7).

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 6-7 pregnant mice were given daily doses of the test substance or the vehicle from day 11 to 17 of gestation via s.c. injection. On day 18, the animals were sacrificed, and fetuses were removed surgically from mothers. Fetal skeletons were examined by means of alcian blue an alizarin red S stained preparation and histologically. The number of live/dead fetuses and the body weight of live fetuses was examined.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

other: QDJ:DDD

Route of administration:

subcutaneous

Vehicle:

not specified

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data

Duration of treatment / exposure:

GD 11-17

Frequency of treatment:

daily

Duration of test:

Seven days

No. of animals per sex per dose:

6/7

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Abnormalities:

not specified

Developmental effects observed:

not specified

MATERNAL TOXIC EFFECTS BY DOSE LEVEL AND BY SEX
No effects reported.

FETAL DATA
Fetal weight significantly (P<0.005) decreased by 44% in test group (mean bw = 0.70 g) versus controls (mean bw =1.26 g). 
Note: lower body weight may reflect larger litter size in treated group (not discussed by authors): 
Controls: 6 dams, 31 live young, 10 resorption sites
HEDP-treated: 7 dams, 52 live young, 18 resorption sites

Alcian blue / Alizarin red S staining demonstrated clear skeletal effects:
- shortening of mineralization zones  and angulation deformities present in long bones, especially femur, tibia, humerus, radius, ulna and rib;
- ossification defects
- calcification of cartilaginous skeleton 
Note: only qualitative data presented

Conclusions:

The described effects can not be excluded at exaggerated doses, and would not be in contrast to the proposed mode of action for general systemic effects at high doses, namely the complexation of essential metals (calcium deficiency). Nevertheless, the outcome of the study can not be considered, mainly because maternal toxic effects were not assessed, and because the chosen route (s.c. injection) was inappropriate to assess the effects after oral, dermal or inhalation exposure. Furthermore, it was not the intention of the study to derive NOAEL or LOAEL values.

Executive summary:

Groups of 6-7 pregnant mice received either a dose of 200 mg/kg disodium HEDP or saline (vehicle control) during GD 11-17 by sucbutaneous injections. On day 18, the dams were sacrificed and fetal skeletons were histologically analyzed by means of alcian blue and alizarin red S staining. Furthermore, the number of live/dead fetuses and the body weight of each fetus were examined. No information on maternal toxicity and no other information of fetal parameters are given in the publication.

The authors observed significantly reduced body weights of the live fetuses and a disturbance of bone development. The decrease in body weight of live fetuses can e.g. be a secondary effect caused by a decreased body weight of the dams. In consideration of the high dose applied, it is likely, but was not examined. Another explanation for the reduced body weights of the fetuses would be the higher number of fetuses counted in the dose group (7.4 per dam) compared to the control group (5.2 per dam). However, in both groups the number of fetuses seems to be low.

The impact on bone development in the offspring might be triggered by maternal toxic effects, too. Based on the assumption that gastrointestinal absorption of orally administered HEDP would occur at a rate of <=10% (see summary of toxicokinetics), a subcutaneous dose of 200 mg/kg bw would correspond to an exaggerated oral dose of ca. 2000 mg/kg bw, which is ten times higher than the expected maternal toxic dose. Furthermore, no first pass effect in the liver occurs after subcutaneous administration.

Reference 8

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

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

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.

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

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

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.

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).

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.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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

 

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.

Reference 9

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

21.05.1980 to 09.06.1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The restrictions were that there was no analytical confirmation of dosing solutions, and there was no record of gravid uterine weight or the number of corpora lutea.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

No record of gravid uterine weight, number of corpora lutea not recorded, no analytical confirmation of dosing solutions.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: No data
- Weight at study initiation: 180-200 g
- Fasting period before study: No data
- Housing: Individually in suspended stainless steel mesh cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: None, animals were recieved mated and allocated to cages.


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ±2
- Humidity (%): 40-60
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26.05.1980 To: 09.06.1980

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution

Details on exposure:

No data

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data. Animals were received on gestational day 1, having already been mated.

Duration of treatment / exposure:

GD 6 - 19

Frequency of treatment:

daily

Duration of test:

20 days

Dose / conc.:

500 mg/kg bw/day

Remarks:

neutral sodium salt; expressed as active acid

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

neutral sodium salt; expressed as active acid

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

neutral sodium salt; expressed as active acid

No. of animals per sex per dose:

25

Control animals:

other: 0.9% (w/v) aqueous NaCl

Details on study design:

- Dose selection rationale: No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for visible toxic effects.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Gestational days 3, 6, 8, 10, 13, 15, 17 and 20.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross examination of all animals that included examination of external surfaces and thoracic and abdominal cavities.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

Comparison of body weights between treatment and control groups was performed using Dunnett's test. Counted data (corpora lutea, implants, resorptions, live and dead fetuses) and data expressed as percentage were analysed, when appropriate with the Mann-Whitney U test. Response data (pregnancy rates, number of litters with post-implantation loss, and fetuses or litters with abnormalities and variants) were analysed, when appropriate, with Fisher's exact test and the chi-square test.

Indices:

None

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No deaths occurred prior to the scheduled sacrifice. 9/25 dams had soft stools in the 2000 mg/kg bw/day group beginning on gestation day 14 (ninth day of treatment) and persisted through to gestation day 17. This finding was not observed in the other treated groups or the controls. The only statistically significant (P<0.01) effect observed was lower body weight gain (mean value approximately 68% of the control mean) between gestation day 6 and 20 for dams in the 2000 mg/kg bw/day group (33.6/27.6/27.5/22.9). However, terminal body weights were not statistically significantly affected (mean terminal body weights with uterine contents by dose: 360.1/368.1/357.0/346.1 and mean terminal bodyw eights without uterine contents: 265.8/264.5/260.1/257.2). There was no effect on pregnancy rate or the mean number of corpora lutea. There were no treatment-related findings in the gross necropsy.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no significant differences between any of the treatment groups and the control group in the number of pre- or postimplantation losses, rresorptions or mean number of live fetuses. There were no effects on fetal body weight, sex ratios (males/litter: 5.6/5.8/5.9/4.8. Females/litter:  5.9/6.5/5.4/6.4). There were no treatment-related gross external malformations present. Subcutaneous haematomas were present in controls and all treated groups, however the incidence increased at 500 mg/kg bw/day (P<0.05) and was considered unrelated to treatment by the study authors (no dose relationship was present). The occurence of one female fetus that was hydrocephalic and one female with gastroschisis in the 1000 mg/kg bw/day group was considered by the study authors to be spontaneous. The hydrocephalic female also exhibited a developmental variation (underdeveloped renal papilla) that was not considered to be related to treatment.
Skeletal examination revealed single incidences of dwarfism (one female fetus of 500 mg/kg bw/day group) and fused sternebrae (one female fetus of the 2000 mg/kg bw/day group), which were considered spontaneous. Two fetuses from different litters in the 2000 mg/kg bw/day group and one fetus of the 1000 mg/kg bw/day group had vertebral anomalies (missing, reduced or fused vertebral arches). Although the incidence of these anomalies was not statistically significant compared with the control group, the rare spontaneous occurrence of such anomalies and the pattern of incidence indicated that they might have been treatment-related. Various skeletal variations occurred, but none showed a clear dose-response and so were considered spontaneous. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a good quality prenatal developmental toxicity screening study (reliability score 2) conducted using a protocol similar to OECD 414, and to GLP, clear maternal toxicity (approx. 30% decrease in body weight gain, soft stools) was noted in pregnant SD rats given 2000 mg/kg bw/day DTPMP (Dequest 2061; expressed as active acid) on GD 6-19 (NOAEL 1000 mg/kg bwt/d). The NOAEL for developmental toxicity was 2000 mg/kg bw/day (active acid).

Executive summary:

In a good quality prenatal developmental toxicity screening study (reliability score 2) conducted using a protocol similar to OECD 414, and to GLP, mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/kg bw/day (neutral sodium salt; expressed as active acid). They were dosed on gestation days 6 to 19. Control animals were given 0.9% sodium chloride solution. On day 20 all surviving animals were sacrificed. There were no deaths prior to scheduled sacrifice. The highest dose produced marginal toxic effects in the dams, as evidenced by lower mean body weight gain between gestational days 6 and 20, and soft stools in some animals. No treatment-related lesions were detected at gross necropsy of the dams of any treatment group.

There were no statistically significant treatment-related effects on postimplantation loss or fetal weight at any dose. Vertebral anomalies were observed in single fetuses in two litters of the 2000 mg/kg bw/day group and one of the 1000 mg/kg bw/day group. The incidence was not statistically significant, but the rare spontaneous occurrence of this type of malformation and the pattern of incidence was suggestive of a treatment-related effect. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity.

Overall, the NOAEL for maternal toxicity was 1000 mg/kg bw/day, and for developmental toxicity was 2000 mg/kg bw/day (active acid).

Reference 10

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

The in-life phase of the study was conducted between 13 June 2012 (first day of treatment) and 04 August 2012 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Harlan Laboratories U.K. Ltd.
- Age at study initiation: approximately twelve weeks old.
- Weight at study initiation: At the start of treatment the males weighed 306 to 351g, the females weighed 189 to 219g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C
- Humidity (%): The relative humidity control was set to achieve target values of 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Distilled water. Formulations were prepared fortnightly and stored at approximately 4ºC in the dark.

All formulation were adjusted for 10.95% water content. All dose levels of expressed in terms of Active Ingredient

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken and analysed for concentration of test item.

The concentration of test item in the test item formulations was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) using an external standard technique.

The results indicate that the prepared formulations were within ± 3% of the nominal concentration.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation).

Frequency of treatment:

The test item was administered daily.

Duration of test:

Up to eight weeks.

Dose / conc.:

100 mg/kg bw/day

Remarks:

active ingredient adjusting for 10.95% water content

Dose / conc.:

350 mg/kg bw/day

Remarks:

active ingredient, adjusting for 10.95% water content

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

active ingredient, adjusting for 10.95% water content

No. of animals per sex per dose:

10 males and 10 females per dose (including control)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were chosen based on the results of a 7-day range-finding study.

Maternal examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
- Motor Activity
- Forelimb/Hindlimb Grip Strength
- Sensory Reactivity


BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

REPRODUCTIVE SCREENING:
Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skin (hind limb)
Eyes
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

All tissues were despatched to the histology processing Test Site for processing.
Microscopic examination was conducted by the Study Pathologist

Ovaries and uterine content:

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

Fetal examinations:

Not examined.

Statistics:

Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Historical control data:

Normal range data for the different parameters examined were used in comparison against the test data.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
There were no unscheduled deaths.

CLINICAL OBSERVATIONS:
No clinical signs of toxicity were detected in any treated animal.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

FUNCTIONAL PERFORMACE TEST:
There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODYWEIGHT:
There were no toxicologically significant effects detected in body weight development.

FOOD CONSUMPTION:
No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

WATER CONSUMPTION:
Daily visual inspection of water bottles did not reveal any significant intergroup differences.

REPRODUCTIVE PERFORMANCE:
Mating:
There were no treatment-related effects on mating performance.

Fertility:
There were no treatment-related effects on fertility.
One female treated with 100 mg/kg bw/day A.I. was non-pregnant. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of this missing pregnancy.

Gestation Length:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 23½ days.

HAEMATOLOGY:
There were no toxicologically significant effects detected in the haematological parameters examined.

BLOOD CHEMISTRY:
There were no toxicologically significant effects detected in the blood chemical parameters examined.

PATHOLOGY:
NECROPSY:
Adults:
No toxicologically significant macroscopic abnormalities were detected.

ORGAN WEIGHTS:
No toxicologically significant effects were detected in the organ weights measured.

HISTOPATHOLOGY:
No treatment related microscopic findings were detected.

Dose descriptor:

NOEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Remarks on result:

other: embryonic/teratogenic effects were not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

Litter Responses:

In total nine females from control and 100 mg/kg bw/day A.I. dose groups and ten females from 350 and 1000 mg/kg bw/day A.I. dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:

No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Pre and post implantation losses were increased in all treated groups when compared to control females. Statistical analysis of the data did not reveal any significant intergroup differences and a true dose related response was not evident. The number of corpora lutea, implantation sites and number of offspring born were also comparable to control females therefore the intergroup differences were considered not to be of toxicological importance.

Offspring Growth and Development:

There were no toxicologically significant effects detected.

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Pathology:

Necropsy:

Offspring

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Conclusions:

The oral administration of [[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt to rats by gavage, at dose levels of 100, 350 and 1000 mg/kg bw/day A.I., did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day A.I., equivalent to 800 mg/kg bw/day active acid.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day A.I.

Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Ha:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 350 and 1000 mg/kg bw/day A.I. (adjusting for 10.95% water content). A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Adult Responses:

Mortality.There were no unscheduled deaths.

Clinical Observations. No clinical signs of toxicity were detected in any treated animal.

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Body Weight. There were no toxicologically significant effects detected in body weight development.

Food Consumption. No adverse effect on food consumption or food efficiency was detected in treated animals.

Water Consumption. No adverse effect on water consumption was detected.

 

Reproductive Performance:

Mating. There were no treatment-related effects on mating for treated animals.

Fertility. There were no treatment-related effects on fertility.

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

 

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

 

Laboratory Investigations:

Haematology. There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry. There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Pathology:

Necropsy. No toxicologically significant macroscopic abnormalities were detected.

Organ Weights. No toxicologically significant effects were detected in the organ weights measured.

Histopathology. No treatment related microscopic abnormalities were detected.

 

Conclusion.

The oral administration of [[(2-hydroxyethyl)imino]bis(methylene)]-bisphosphonic acid, equilibrium mixture with 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium saltto rats by gavage, at dose levels of 100, 350 and 1000 mg/kg bw/day A.I., did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day A.I.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day A.I.

Reference 11

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only a summary report was available for review.

Principles of method if other than guideline:

This is a non-standard study to investigate the teratogenic potential of HEDP.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles River CD

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

No details available.

Duration of treatment / exposure:

Various (see below)

Frequency of treatment:

Continuous

Duration of test:

No data. Pups were sacrificed approximately 56 days after weaning.

No. of animals per sex per dose:

No data

Control animals:

yes, plain diet

Dose descriptor:

NOAEC

Effect level:

>= 0.5 other: % in diet

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

>= 0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Abnormalities:

not specified

Developmental effects observed:

not specified

One female in Group C was necropsied prematurely because of a neck tumour. No unusual lesions were noted grossly at the scheduled necropsies. Apart from the neck tumour, no other unusual lesions or changes were observed microscopically. There were no histological differences between the test and control animals in either the F0 or the F1 generations. No further details available.

Conclusions:

In a non-standard dietary teratology study, only available as a brief summary, and not to GLP (reliability score 4) the NOAEC for HEDP was at least 0.5% HEDP in diet in rats.

Executive summary:

In a non-standard dietary teratology study, only available as a brief summary, and not to GLP (reliability score 4) the NOAEC for HEDP was at least 0.5% HEDP in diet in rats.

Additional information

The two key studies with HEDP salts which were published in peer-reviewed literature (Nolen & Buehler, 1971) include data on two species (rats, rabbits) and two different study designs (two-generation study, teratogenicity). In general, the tests meet the scientific principles of the corresponding OECD guidelines No. 416 and No. 414. The deviations relevant for the assessment of developmental toxicity have been examined. Though only two doses were tested instead of three, the dose selection can be considered appropriate to evaluate the teratogenic potential, as maternal toxic effects were induced at the highest dose. In the developmental toxicity study with rabbits, the dosing took place from day 2-16 instead of day 6-18. This time span was considered best practice at the time the test was performed. In spite of deviations from the guidelines, adequate information is provided in the two key studies to conclude that HEDP and its salts do not produce any adverse on developmental toxicity in two species (rat, rabbit). Details of the two studies are discussed below.

 

In a pre- OECD combined two-generation study of reproductive toxicity and teratogenicity, five groups of 22 female and 22 male weanling Charles-River rats were given the disodium salt of HEDP (disodium etidronate) at a dietary concentration of 0, 0.1 or 0.5% (equivalent to a dose of 0, 112 or 447 mg/kg bw per day), either continuously or only on days 6–15 of gestation for two generations. Reproductive endpoints and offspring parameters were analyzed in the F1a, F1b, and F2a litters. The third litter of the F1 generation (F1c) and the second litter of the second generation (F2b) were used in teratological examinations. During the teratology phase, half of the animals in each group were sacrificed at day 13 and the others at day 21 of gestation. Body-weight gains were similar for all groups in both generations, and the overall conception rate was 90%, indicating that the compound did not interfere with spermatogenesis or ovulation. In the first generation, at the highest dose, the number of pubs born in the first litter (F1a) was reduced and there was an increase in stillborn pups in the second litter (F1b). The rate of mortality in pups after birth was low and the weights of pups at day 4 and at weaning were the same. Teratological examination of the third litter (F1c) showed no differences in resorptions or implantations in females sacrificed at day 13 and no differences in live fetuses, corpora lutea, or implantation at 21 days. At day 21, however, significant resorptions were reported in the controls. In the second generation, the first litters (F2a) were smaller than the litters in the first generation, but there were no other differences in reproductive parameters. During the teratology phase, no differences in corpora lutea, implantations, or resorptions were noted in rats sacrificed at 13 days. In continually fed rats sacrificed at 21 days, the number of implantations was reduced and corpora lutea formation was depressed at the highest dose. A decrease in the number of live fetuses at the highest dose, significant only in rats fed during gestation, was also observed. The incidence of defective pups was similar to that in control animals.

The NOAEL for maternal toxicity was set at 112 mg/kg bw per day (Nolen & Buehler, 1971). The study authors concluded that disodium etidronate was not teratogenic in rats at either dose tested (NOAEL developmental toxicity: >= 447 mg/kg bw per day).

 

A preparatory study inrabbits (Nolen and Buehler, 1971) found that doses of 500 mg/kg bw/day in water by intubation induced maternal death, so the doses were reduced to 250 mg/kg bw/day in four surviving animals for the rest of the study. The results show that during pregnancy rabbits are more sensitive than rats towards maternal toxicity and that - based on a limited number of remaining maternal animals - a dose of 250 mg/kg bw/day is a potential NOAEL for developmental toxicity in rabbits. In the subsequent feed study no evidence of maternal or fetal effects was apparent when rabbits were fed diets designed to deliver the equivalent of 25, 50 or 100 mg disodium etidronate/kg bw/day from day 2 through to day 16 of pregnancy (Nolen and Buehler, 1971). There were no apparent treatment-related effects on pregnancy rates, numbers of corpora lutea, implantations, resorptions or live fetuses and their weights, in either of the studies in rabbits. Nor was there a treatment-related increase in teratogenic effects observed in either study. a reduction in fetal weights with HEDP at a dose of 100 mg/kg bw per day administered by gavage, was attributed by the study authors to slightly larger litters. The combined results from the preparatory study and the main study indicate that the NOAEL for maternal toxicity in the rabbit is at least 100 mg disodium etidronate/kg bw/day. The NOAEL for developmental toxicity was concluded to be >= 100 mg/kg bw/day on the basis of 886 rabbit fetuses examined in the main study.

 

Two publications on sodium salts of HEDP (King, 1967 and Eguchi, 1982) are not considered for the derivation of a NOAEL for developmental toxicity because of missing background information and methodological deficiencies. However, the information given in those publications was evaluated within a weight-of-evidence approach.

 

Further evidence for the assessment of developmental toxicity is provided by a study with only few data available on the potential impact of the parent etidronic acid on fetal development (Monsanto plc 1966). 20 female Long-Evans rats per dosing group were tested according to the FDA guidelines for reproduction studies for safety evaluation of drugs for human use. They were given 16.5, 110 or 330 mg/kg bw/d by gavage, 2 times daily half of the total daily dose, during days 6 -15 of gestation. For all test material concentrations no effect was observed, therefore a NOAEL => 330 mg/kg bw/d was concluded.

 

In a non-standard dietary teratology study (King, 1967), only available as a brief summary, the NOAEC for HEDP was at least 0.5% HEDP in diet in rats.

 

Eguchi et al. (1982) reported significantly reduced body weights of the live fetuses and a disturbance of bone development in female mice administered HEDP by subcutaneous injection at 200 mg/kg bw/d on gestation days 11-17.

Having regard to likely (non medical) routes of human exposure subcutaneous injection is not an appropriate route of administration. The effects might be very well triggered by maternal toxic effects although an examination of maternal toxicity seemed not to be part of the Eguchi study. Based on the assumption that gastrointestinal absorption of orally administered HEDP would occur at a rate of <= 10% (see summary of toxicokinetics), a subcutaneous dose of 200 mg/kg bw would correspond to an exaggerated oral dose of ca. 2000 mg/kg bw, which is much higher than the expected maternal toxic dose for mice. Additionally, no first pass effect in the liver occurs after subcutaneous administration and differences in the disposition of HEDP are likely related to the uncommon route of administration. Nevertheless, the described effects can not be excluded at exaggerated doses, and would not be in contrast to the proposed mode of action for general systemic effects at high doses, namely the complexation of essential metals leading e.g. to calcium and iron deficiency. Another explanation for the reduced body weights of the fetuses would be the higher number of fetuses counted in the dose group (7.4 per dam) compared to the control group (5.2 per dam). Last but not least, single treatment level, use of a non preferred rodent species, low number of animals (control: 6, treatment group 7), and generally poor reporting of methods and results impose limitations on the overall suitability of the study for a quantitative risk assessment.

  

Several other results from guideline developmental toxicity studies in rats and mice performed using structurally-related phosphonic acid analogues (ATMP and salts; HEBMP salt, DTPMP and its salts) are included in the dossier as part of the weight of evidence approach. The related phosphonic acid compounds from the ATMP, HEBMP, and DTPMP categories are not teratogenic following oral treatments between 312 and >= 1000 mg/kg bw/day during pregnancy.

 

Based upon these considerations, a developmental NOAEL of >= 100 mg/kg bw/day appears appropriate for HEDP and its salts.

Justification for grouping:

See CSR Annex I and II or IUCLID section 13.

Justification for classification or non-classification

No classification is proposed for the reproductive and developmental toxicity of tetrasodium HEDP.