Tetrasodium (1-hydroxyethylidene)bisphosphonate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No guideline available, non-GLP study.

Qualifier:

no guideline available

Principles of method if other than guideline:

In a 28-day feeding study with juvenile rats, the effect of HEDP on bone resorption was evaluated.

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

other: effect on bone resorption

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- juvenile Wistar - SPF - rats (male and female)
- recieved from Mus Rattus, Brunnthal, on 03.07.1981
- age: approx. 4 weeks old
- mean bodyweight: 97g at beginning of the study
- acclimatization time: 3 days
- identification: color-coded cards on the cages; individuals characterized by specific color-codes on the fur (coloring with picric acid acc. Teuffel)

ENVIRONMENTAL CONDITIONS
- hygiene: conventional conditions
- housing: 2-3 males or 5 females in Makrolon-cages type 3
- bedding: autoclaved softwood granulate
- temperature: 21°C +/- 2°C
- relative humidity: 50% +/- 5%
- 12h of artificial light per day
- food: Altromin 1324 DK (Altromin GmbH; low-germ, low in nitrosamine pellets), ad libitum
- water: tap-water, ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

During the study, the food-preparations were stored under laboratory's conditions. They were formerly prepared by Altromin Spezialwerke GmbH using a triturate process to mix the test material into the food. The respective doses of 500 / 2000 / 10000 ppm of HEDP were recieved by using 638 / 2551 / 12755 mg HEDP (78.4%) per kg rat food. The final food was administered ad libitum.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 500, 2000, 10000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0 / 48.5-50 / 203.9-190.5 / 965 -1135.2 mg/kg bw/d
Basis:
nominal in diet
calculation based on mean bw (m/f) and active acid

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Examinations:

EXAMINATIONS DURING THE STUDY:
- mortality: daily (twice)
- symptoms of intoxication: daily (twice)
- bodyweight: weekly
- food consumption: weekly
- differential blood count: after 4 weeks of application (end of study)

EXAMINATIONS AFTER 28 DAYS:
- haematology
- pathology
- histology
- effects on bones (tibia, metatarsus, calcaneus)

GENERAL OBSERVATIONS, FOOD CONSUMPTION AND BODYWEIGHT

All rats showed the same normal accepting behaviour with regard to the food compared to the controls.

The complete study was proceeded without any abnormalities.

All animals of the controls and the respective dosing groups survived the 28 day period of the study. No substance-related symptoms could be recorded during that time.

dosing group sex	1 control		2 500 ppm		3 2000 ppm		4 10000 ppm
	m	f	m	f	m	f	m	f
mean food intake (g/kg bw/d)	113.7	105.9	101.0	97.0	102.0	95.3	113.5	96.5
mean HEDP intake (mg/kg bw/d)	-	-	50.5	48.5	203.9	190.5	1135.2	965.0
mean bodyweight increase (g)	170	89	178	86	180	94	181	106

 

In general, the mean food consumption especially in males was slightly decreased compared to the controls.

In the HEDP dosing groups, a slightly higher mean bodyweight increase was observed compared to the controls.

 

HAEMATOLOGY

The differential blood count was examined microscopically.

1 male individual of group 2 (500 ppm) exhibited a high increase of banded neutrophil granulocytes, in 1 male individual of group 4 (10000 ppm) a moderate increase of banded neutrophil granulocytes was observed.

 

PATHOLOGY AND HISTOLOGY

In all dosing groups, including control, hydronephrosis of the kidneys was observed to variant degrees. In some females, the presence of hydrometra was found. Beside that, all other organs only showed species-typical individual results independent from dosing and without any noticeable accumulations.

 

MORPHOMETRY

In the highest dosing group (10000 ppm) an enlargement of the tibiae could be observed to a low degree.

 

GRAVIMETRY

Following HEDP application, an increase of the absolute and relative weights of the tibiae compared to the controls was found. This observation was relevant for all HEDP dosing groups and not dependent on the amount applied and could be interpreted as an inhibitory effect on the bone resorption. This result was more obvious for males than for females.

All bones of controls and dosing groups were without any findings in the x-ray radiographs.

 

Absolute weights of the tibiae

 

group	# males	Mean weight (g)	SD	# females	Mean weight (g)	SD*
Control	5	0.2553	0.0064	5	0.2107	0.0095
500 ppm	5	0.2887	0.0163	5	0.2372	0.0231
2000 ppm	5	0.2887	0.0103	5	0.2299	0.0260
10000 ppm	5	0.2911	0.0162	5	0.2370	0.0138

 

Relative weights of the tibiae

 

group	# males	Mean weight (%)	SD	# females	Mean weight (%)	SD*
Control	5	0.1019	0.0013	5	0.1230	0.0052
500 ppm	5	0.1069	0.0063	5	0.1359	0.0101
2000 ppm	5	0.1045	0.0041	5	0.1226	0.0049
10000 ppm	5	0.1057	0.0019	5	0.1217	0.0043

 

*SD: standard deviation

Conclusions:

Application of the high dose of approx. 965-1135 mg/kg bw/d led to low-degree enlargement of the tibiae. Dose-undependent increases in the absolute and relative weights of the tibiae were found at all dose levels.

Executive summary:

In a 28-day feeding study, rats received doses of 0/500/2000/10000 ppm of HEDP in the diet (corresponding to appr. 0/48.5-50/203.9-190.5/965 -1135.2 mg/kg bw/d). Besides the evaluation of mortality, symptoms of intoxication, body weights, food consumption, haematology and (histo-)pathology, specific examinations of the three bone types (tibia, metatarsus, calcaneus) were conducted.

In the highest dose group, the application of HEDP led to a low-degree enlargement of the tibiae. Dose-independent increases in the absolute and relative weight of the tibiae, but not of the other examinated bones, were found at all dose levels. However, the findings did not correspond to the x-ray examinations, that did not exhibit any alterations. No adverse effects were observed with regard to any other of the investigated parameters. The study directors concluded that the changed in tibia weights might be interpreted as inhibitory effect on bone resorption.

The study focused on medical aspects rather than on toxicological assessment. With regard to the latter, the study supports the presumption that HEDP can act as a complexing agent. This mode of action was also observed in toxicological studies summarized in chapter 7.5 (repeated dose toxicity). At the lowest dose of 48.5-50 mg/kg bw/day, which corresponds to the NOAEL derived in the repeated dose toxicity key study, marginal effects on the tibia weights were observed. However, it remains unclear, whether these effects could be interpreted as adverse effect, or as a positive impact on the prevention of bone resorption. The study directors did not deduce a NOAEL, which was probably not in the scope of the study.

Description of key information

Two studies with pharmaceutical background, focusing on the effects on bones, revealed marginal increase in bone growth. These effects are in line with the postulated effect that HEDP acts as a complexing agent for metals such as calcium. However, in the absence of an adverse effect and the limited number of organs examined, the studies do not allow the derivation of a NOAEL value.

Additional information

Numerous abstracts/citations addressing the potential use of HEDP in cancer therapy, osteoporosis, Paget disease, nuclear imaging, and hypercalcaemia associated with malignancy, and other disorders of calcium and phosphorus balance have been publishedin the pharmaceutical literature.

Two rat studies with medical background conducted with sodium salts of HEDP, for which complete study reports are available, are described in more detail in the endpoint study records of this chapter. Both studies investigated medical rather than toxicological parameters in rats after oral application of 28 days or 2 years. The reliability with regard to the toxicological impact was not assignable (Klimisch 4), allowing no deduction of a NOAEL.

In the study with subacute application, the high dose of approx. 965-1135 mg/kg bw/d led to low-degree enlargement of the tibiae. In the absence of morphological changes, only dose-independent increases in the absolute and relative weights of the tibiae, but not of other examined bones, were found at all dose levels. All other investigated parameters including X-ray analyses were not affected.

In the 24 month-study, groups of rats (150 per group) were treated with either 14C-HEDP (3.3 ppm) or NaF (3.3 ppm) in their drinking water, controls (2 groups of 75 animals per group) received untreated drinking water. The mean daily intake of HEDP was 0.184 mg/kg bw. Only a minimal amount of HEDP was found in the bone, and compared to NaF, the accumulation of HEDP was significantly less. Numerous additional evaluations including biochemical parameters of bone metabolism, X-ray studies and bone-morphometry showed no treatment-related adverse effects of HEDP.

Both studies focused on the effects on bones as target organs. The marginal effects observed are in line with the postulated effect that HEDP acts as a complexing agent for metals such as calcium. However, in the absence of an adverse effect and the limited number of organs examined, the studies do not allow the derivation of a NOAEL value.