(1-hydroxyethylidene)bisphosphonic acid, potassium salt

Endpoint summary

Description of key information

There are no repeated dose toxicity data for HEDP-xK, therefore good quality data are read-across from the category member HEDP (2-3Na).

In a well conducted, repeated oral dose toxicity study (reliability score 2), conducted using a protocol according to OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) and pre-GLP, the NOAEL for HEDP (2-3Na) was concluded to be 41 mg/kg bw/day (equivalent to 34 mg active acid/kg bw/day) based on anaemia at high doses (Huntingdon, 1977).

No repeated dose inhalation or dermal studies are available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.07.1976 to 17.07.1978 (for full study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Conducted prior to guideline adoption.

GLP compliance:

no

Remarks:

pre-GLP

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 grams
- Fasting period before study: No
- Housing: Five per cage in suspended cages with wire-mesh floors.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Six days


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


IN-LIFE DATES: From: 19.07.76 To: 17.07.78

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

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

Analytical verification of doses or concentrations:

no

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. Therefore, dietary concentrations not verified during interim study period.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Dose / conc.:

500 ppm

Remarks:

males: 41 mg/kg bw/day
females: 50 mg/kg bw/day (calculated by the reviewer)

Dose / conc.:

2 000 ppm

Remarks:

males: 169 mg/kg bw/day
females: 195 mg/kg bw/day (calculated by the reviewer)

Dose / conc.:

10 000 ppm

Remarks:

males: 817 mg/kg bw/day
females: 1000 mg/kg bw/day (calculated by the reviewer)

No. of animals per sex per dose:

Ten

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment: 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 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: No data

Positive control:

None

Observations and examinations performed and frequency:

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.

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 groups; Week 12 from 10 males and females in all groups; Weeks 25 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 and 25 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 and 25: 5 males and 5 females from control and 10000 ppm; 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, 2000 ppm and 10000 ppm for glucose and serum alkaline phosphatase. Week 12: 5 males from 500 ppm and 2000 ppm for serum alkaline phosphatase and serum glutamic pyruvic transaminase. Week 13: 5 females from all groups - plasma glucose. Week 25: 5 males from 2000 ppm group for serum alkaline phosphatase and 5 females from 2000 ppm group for plasma glucose.
- Animals fasted: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 6 and 25: 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1)
HISTOPATHOLOGY: Yes (see Table 1)

Other examinations:

None reported

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

Description (incidence and severity):

Severe pallor of skin in rats receiving 10 000 ppm and slight pallor in rats receiving 2000 ppm from week 6 were observed.

Mortality:

no mortality observed

Description (incidence):

Mortality occurred in one control female animal under anaesthesia for blood collection.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first 12 weeks of treatment, a significantly reduced body weight gain was recorded for the 10 000 ppm group. There were no other statistically significant differences between the control and treated groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was marginally (but statistically significant) reduced in highest dose group male rats. All other groups as for controls.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect was observed in any of the dose groups.

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

no effects observed

Description (incidence and severity):

In the highest dose groups, a reduced water consumption was observed which was in line with the reduced food consumption

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During weeks 5 and 7, the 10000 ppm group had a statistically significant decrease in red cell parameters. Neutrophil and lymphocyte counts were significantly higher than controls. Additionally, a decrease in red cell values and higher neutrophil and lymphocyte counts for 2000 ppm males at week 7 (not studied at week 5) were observed. No similar statistically significant difference occurred for females. During week 12, a reduction in red cell parameters for both sexes at 10 000 ppm and for males at 2000 ppm was noticed. Moreover, a slightly higher platelet count for the 10000 ppm male group was observed. Examination of blood smears indicated a retardation of bone marrow development and prolonged anaemia at weeks 5, 7, 12 in both sexes at 10 000 ppm. During week 7, a slight retardation was seen in the 2000 ppm male group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher alkaline phosphatase at week 5, 7 and 12 in males receiving 10 000 ppm was observed. No effects were observed in the 2000 ppm dose group at week 7. All other parameters were similar in control and treated males. Higher plasma glucose level in females receiving 10000 ppm at week 12 and 13 was observed, however, no effects in the lower dose females occurred.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary volume was greater in the male treated groups at weeks 6 and 7, however, there were no differences at week 12. Non-dose-related sporadic increases in pH were detected in some rats, although this was not considered as of toxicological significance. Increased calcium in highest dose males at weeks 6 and 12 were reported, however, only when expressed in terms of volume of urine and not in absolute terms.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant decrease in liver weights recorded for males and females in the 10 000 ppm group. At 2000 ppm, the effect was observed in males, although at a significantly smaller magnitude. Additionally, a statistically significant decrease in kidney weight in the highest dose males was observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings of toxicological significance was observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related findings.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The bone marrow smears revealed a decrease in myeloid/erythroid and lymphocyte/erythroid ratios for rats in the highest dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: Effect level refers to disodium salt; equivalent to 34 mg/kg bw/day active acid

Sex:

male

Basis for effect level:

other: juvenile rats (500 ppm of HEDP (2-3Na)

Dose descriptor:

LOAEL

Effect level:

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

Based on:

other: Effect level refers to disodium salt; equivalent to 139 mg/kg bw/day active acid

Sex:

male

Basis for effect level:

other: anaemia (2000 ppm HEDP (2-3Na)

Critical effects observed:

not specified

Conclusions:

In a well conducted, repeated oral dose toxicity study (reliability score 2), conducted using a protocol according to OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) and pre-GLP, the NOAEL for HEDP (2-3Na) was concluded to be 41 mg/kg bw/day (equivalent to 34 mg active acid/kg bw/day) based on anaemia at high doses.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

34 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

(active salt)

Organ:

blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a well conducted, repeated oral dose toxicity study (reliability score 2), conducted using a protocol according to OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) and pre-GLP, ten male and ten female Sprague-Dawley rats were fed 0 ppm, 500 ppm, 2000 ppm or 10 000 ppm (corresponding to 0, 41, 169 or 817 mg/kg bw/day for males and 0, 50, 195 and 1000 mg/kg bw/day for females) of HEDP (2-3Na) for 90 days.

A decrease in the red blood cell parameters was observed in the highest dose group for both sexes as well as at the mid-dose for males by week 12, although some improvement was noted from the week 7 values. Blood smears indicated prolonged anaemia in both sexes at the highest dose, with a slight retardation of bone marrow development. In the mid-dose, slight pallor was observed and severe pallor of the skin in the highest dose group. Additionally, a pale colour was noted for organs well supplied with blood (spleen and kidneys) which are consistent with perturbation of iron homeostasis. During week 25, values relating to red cell parameters among animals receiving 2000 ppm were similar to control values. For animals receiving 10 000 ppm, the packed cell volume and haemoglobin concentrations were only marginally lower, although statistically significant compared to the control group. It was concluded that the NOAEL for HEDP-2Na in juvenile rats is 500 ppm (equivalent to 41 mg/kg bw/day or 34 mg active acid/kg bw/day) in males based on anaemia at high doses (Huntingdon, 1977).

In a dietary Combined Chronic Toxicity / Carcinogenicity study (reliability score 2), conducted using a protocol similar to OECD Test Guideline 453 and pre-GLP, the NOAEL for HEDP (2-3Na) was 78 mg/kg bw/day for males and 96 mg/kg bw/day for females, based on anaemia at higher doses (Huntingdon, 1979).

In a supporting sub-chronic repeated dose toxicity study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 409 and pre-GLP, a NOAEL of ≥1620 mg/kg bw/day (active acid) in female dogs and a NOAEL of ≥1746 mg/kg bw/day (active acid) in male dogs were determined (Industrial BioTest 1975a).

In a 90-day sub-chronic oral toxicity study (reliability score 2), conducted to a protocol similar to OECD Test Guideline 408 and pre-GLP, HEDP-H (60% active acid) was dosed in concentrations of 0, 3000, 10 000 or 30 000 ppm to rats. The body weight gains for animals in the 30 000 ppm dose group were decreased for both males (84% of controls) and females (92% of controls). The food intake in the 30 000 ppm male dose group was slightly lower (88% of controls) whereas it was normal in the 30 000 ppm female group. Following blood collection, mortality occurred, particularly in the 30 000 ppm dose group (10 animals compared to 1 animal in the control group) suggesting that animals in the higher dose group might have been less able to tolerate the stress of blood collection compared to the control group.

In the 30 000 ppm dose group, increased erythrocyte counts (males), decreased hemoglobin concentration (both sexes), decreased haematocrit values (both sexes), and decreased leukocyte counts (females at 84 days only) were observed. Coagulation time and differential leukocyte counts were not altered in this group. No haematologic changes were observed in the 3000 or 10 000 ppm dose groups. In the 30 000 ppm male group, liver weights were decreased whereas the 30 000 ppm female group as well as all the other dose groups were within the normal range. It is concluded that the NOEL for male and females is >3000 mg/kg bw/day (Industrial Biotest, 1975b).

See read-across justification in IUCLID Section 13.

Justification for classification or non-classification

Based on the available read-across data, adverse effects on iron homeostasis were not considered to be severe enough to trigger classification. Therefore, a classification for STOT-RE is not required for HEDP-xK according to Regulation (EC) No. 1272/2008.