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

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In order to determine the possible cumulative effects of HEDP in rats, a long term study has been performed for two years.
The test animals were treated ad libitum with drinking water containing 14C-HEDP (1-hydroxyethane-l,l-diphorphanic acid? disodium salt) (3.3 ppm).
With respect to the use of HEDP in tooth-paste, the dose level of 3.3 ppm (approximately 0.2 mg/kg bw/day) was selected on the assumption that a certain amount (10%) can be swallowed by using a tooth-paste containing 0.5 - 1% HEDP.
Another test group was treated ad libitum with drinking water containing the same concentration (3.3 ppm) of NaF.
The study enclosed 450 animals. Each of the two groups consisted of 150 test animals and 75 animals in a control group.

GLP compliance:

not specified

Remarks:

pre-GLP

Type of method:

in vivo

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

8 weeks old Wistar male rats. The body weight were approximately 200 g.

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 months

Frequency of treatment:

Continuous

Dose / conc.:

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

Dose / conc.:

3.3 ppm (nominal)

Remarks:

Nominal in water

No. of animals per sex per dose:

150 animals per treatment group, 75 control animals.

Control animals:

yes, concurrent no treatment

Details on study design:

During the whole test period, the daily dose of intake was 0.184 mg of HEDP per kg. This is equivalent to a total intake of 134 mg/kg body weight. In the NaF group, the values were 0.202 mg/kg and day, which amounts to a total intake of 147 mg of NaF/kg body weight.
Every four weeks in 27 series of necropsies the tibiae were prepared. The HEDP content was determined by measuring radioactivity resp. the NaF content by determination of fluoride.

Examinations:

The following evaluations were performed:
macroscopical and microscopical examinations, blood chemistry (including determination of magnesium, iron, zinc in serum), urinalysis, examination of bone marrow smears, Organ weight determination, the determination of calcium and phosphor in trachea and tibia.

Positive control:

No positive control.

Details on results:

Only a small amount of HEDP was found in the bones. Four weeks after the start of the test. the amount of HEDP in the tibiae was 68.5 ppb (68.5 μg of HEDP/kg bone mass) and after 103 weeks, 177 ppb (177 μg of HEDP/kg bone mass).
During this period. the increase of radioactivity in the bones was minimal and almost linear. At this time. the amount of HEDP by drinking water was 0.033 % in the total skeleton and after the test period 0,0065 %.
The bones of the animals in the NaF group showed an increased content of fluoride compared to the control group. The content of fluoride increased linearly during the whole test period. That means NaF and HEDP showed similar biological behaviour in bones.
At the end of the test, X-ray studies showed that there was no influence of HEDP and NaF on morphology and length of the bones.

Macroscopical and microscopical examinations, blood chemistry (including determination of magnesium, iron, zinc in serum), urinalysis, examination of bone marrow smears, organ weight determination, the determination of calcium and phosphor in trachea and tibia did not show any treatment related effects. In both groups, consumption of food and body weight slightly increased compared to the untreated control group.

In autoradiographic studies performed at the end of the test, only weak labelling of the gastrointestinal tract was observed in HEDP-treated animals. This effect was due to the continuous oral intake of 14C-HEDP.

Conclusions:

In a limited summary of a 24 month chronic oral study (reliability score 4),not conducted according to any OECD Test Guideline and pre-GLP, only a minimal amount of HEDP-xNa was found in the bone. At the end of the study, the amount of HEDP-xNa found in tibiae was 177 µg HEDP/kg bone mass, i.e. 0.0065 % of the total intake. Numerous additional evaluations including biochemical parameters of bone metabolism, X-ray studies and bone-morphometry showed no treatment-related adverse effects.

Reference 2

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not specified

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

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)
- source: Recieved from Mus Rattus, Brunnthal, on 03.07.1981
- age: approximatly 4 weeks old
- mean bodyweight: 97 g at the 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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

equivalent to 48.5-50 mg/kg bw/day (nominal) active acid

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 203.9-190.5 mg/kg bw/day (nominal) active acid

Dose / conc.:

10 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 965-1135.2 mg/kg bw/day (nominal) 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)

Details on results:

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

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:

In a 28-day feeding study (reliability score 4), not conducted according to any OECD Test Guideline and not in compliance with GLP, rats received doses of 0, 500, 2000 or 10000 mg/kg bw/day of HEDP-H in the diet (equivalent to 48.5-50, 203.9-190.5 or 965-1135.2 mg/kg bw/day (nominal) active acid. Evaluation of mortality, symptoms of intoxication, body weights, food consumption, haematology and histopathology as well as specific examinations of the three bone types (tibia, metatarsus, calcaneus) were conducted.

In the highest dose group, the application of HEDP resulted in a low-degree enlargement of the tibiae. Dose-independent increases in the absolute and relative weight of the tibiae, however, not of the other examined 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

There are no available key studies on specific investigations for the HEDP category members.

Additional information

In a limited summary of a 24 month chronic oral study (reliability score 4),not conducted according to any OECD Test Guideline and pre-GLP, only a minimal amount of HEDP-xNa was found in the bone. At the end of the study, the amount of HEDP-xNa found in tibiae was 177 µg HEDP/kg bone mass, i.e. 0.0065 % of the total intake. Numerous additional evaluations including biochemical parameters of bone metabolism, X-ray studies and bone-morphometry showed no treatment-related adverse effects (Henkel, 1986).

In a 28-day feeding study (reliability score 4), not conducted according to any OECD Test Guideline and not in compliance with GLP, rats received doses of 0, 500, 2000 or 10000 mg/kg bw/day of HEDP-H in the diet (equivalent to 48.5-50, 203.9-190.5 or 965-1135.2 mg/kg bw/day (nominal) active acid. Evaluation of mortality, symptoms of intoxication, body weights, food consumption, haematology and histopathology as well as specific examinations of the three bone types (tibia, metatarsus, calcaneus) were conducted.

In the highest dose group, the application of HEDP resulted in a low-degree enlargement of the tibiae. Dose-independent increases in the absolute and relative weight of the tibiae, however, not of the other examined 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 (Henkel, 1988).