Tetrasodium (1-hydroxyethylidene)bisphosphonate

Endpoint summary

Description of key information

A NOAEL for HEDP-4Na of 48 mg/kg bw/d was derived based on the lowest NOAEL for HEDP-2Na of 41 mg/kg bw/d for anaemic effects described in the interim report of a combined chronic toxicity / carcinogenicity study with disodium etidronate. The NOAEL takes into consideration the most susceptible life span (juvenile animals).

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:

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 (for full study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according 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

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 oral 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:

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 d

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 (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

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; 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 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, 2000 ppm and 10000 ppm for glucose and serum alkaline phosphatase. Week 12: 5 males from 500ppm 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 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 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

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Mortality: one control female died under anaesthesia for blood collection. Severe pallor of skin in rats receiving 10000 ppm and slight pallor in rats receiving 2000 ppm from week 6.


BODY WEIGHT AND WEIGHT GAIN: During the first 12 weeks of treatment, a significantly reduced body weight gain was recorded for the 10000 ppm group. There were no other statistically significant differences between the control and treated groups.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food intake marginally (but statistically significant) reduced in highest dose group male rats.  All other groups as for controls.


FOOD EFFICIENCY: No effect.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): reduced in highest dose groups in line with reduced food consumption


HAEMATOLOGY: 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. There was also a decrease in red cell values and higher neutrophil and lymphocyte counts for 2000 ppm males at week 7 (not studied at week 5). There were no such statistically significant differences for females. During week 12 there was a reduction in red cell parameters for both sexes at 10000 ppm and for males at 2000 ppm.  Also, 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 10000 ppm. During week 7 a slight retardation was seen in the 2000 ppm male group.


CLINICAL CHEMISTRY: Higher alkaline phosphatase at week 5, 7 and 12 in males receiving 10000 ppm.  No effects seen in 2000 ppm 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.  No effects were observed in the lower dose females.


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


ORGAN WEIGHTS: There was a statistically significant decrease in liver weights recorded for males and females of the 10000 ppm group. At 2000 ppm the effect was observed in males, but at a much smaller magnitude. There was also a statistically significant decrease in kidney weight for highest dose males.


GROSS PATHOLOGY: No findings of toxicological significance.


HISTOPATHOLOGY: NON-NEOPLASTIC: There were no treatment-related findings.


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

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

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

Remarks on result:

other: Effect level refers to disodium salt

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: anaemia (2000 ppm HEDP-2Na)

Remarks on result:

other: Effect level refers to disodium salt

Critical effects observed:

not specified

Conclusions:

In a well conducted and reported, pre-GLP, study (reliability score 2), conducted using a protocol according to OECD 408 (Repeated Dose 90-Day Oral Toxicity in Rodents), the NOAEL for Complexing Agent - Henkel (sodium salt of 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate)) was 41 mg/kg bw/day, based on anaemia at high doses. This study is part of a combined chronic toxicity / carcinogenicity study (equivalent to OECD 453), reported separately in chapter 7.5.1, fist entry.

Executive summary:

In order to reduce the number of animals used, a combined dietary chronic toxicity and carcinogenicity study in rats has been carried out using a protocol similar to OECD 453. Four satellite groups of 10 animals of each sex (dose and control groups) were fed diets containing 0-10000 ppm disodium etidronate and used to provide blood and urine samples during the first 26 weeks of the study. After the first 26 weeks of the study all surviving rats in the satellite groups were killed for interim examination. The results of the interim report for the satellite groups are reported separately because juvenile rats during their growth phase seem to be more susceptible to effects of HEDP related to perturbations of iron homeostasis than adult rats. The doses quoted in the interim report (0, 500, 2000, and 10000 ppm corresponding to 0, 41, 169, 817 mg/kg bw/d for males and 0, 50, 195 and 1000 mg/kg bw/d for females) take into account the higher feed intake as a function of bodyweight during the first few weeks of the study. A decrease in red blood cell parameters was seen in the top dose group for both sexes, and for males at 169 mg/kg by week 12 although some improvement was noted from the week 7 values. Blood smears indicated prolonged anaemia in both sexes at the top dose, with a slight retardation of bone marrow development. Severe pallor of the skin of the top dose group animals and slight pallor in the mid dose rats was seen. A pale color was also noted for organs well supplied with blood (spleen and kidneys). These observations are consistent with perturbation of iron homeostasis. During week 25, values relating to red cell parameters among rats receiving 2000 ppm were similar to control values. For rats receiving 10000 ppm the packed cell volume and haemoglobin concentrations were only marginally lower than control values although the differences attained a level of statistical significance. The NOAEL for juvenile rats is assigned to the lowest dose group (500 ppm) where no indication of anaemia was seen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

41 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

ORAL ROUTE:

In order to reduce the number of animals used, a combined dietary chronic toxicity and carcinogenicity study in rats was carried out (Huntingdon Research Centre, 1979). With the main exception of the number of animals (40 instead of 50 rats of each sex per dose group) the study meets the criteria of the OECD guideline No. 453. Sprague-Dawley rats were fed diets containing 0-10000 ppm disodium etidronate for 104 weeks. The mean doses for the whole two-year study are equivalent to 0, 19, 78 and 384 mg/kg in males and 0, 24, 96 and 493 mg/kg in females. During the study, animals were observed for clinical signs of toxicity and mortality. Observations included body weight, food and water consumption, ophthalmoscopic examination, hematology, clinical chemistry and urine analysis. During the study and at study terminations all animals underwent necropsies and histopathological examinations. No mortality or treatment-related effects were observed on ophthalmoscopy, clinical chemistry or gross pathology. No histopathological changes were found at study termination. A pale colour of the spleen was seen in the mid and high dose group at 26 weeks, indicating a lack of iron. Concordantly, haematological disturbances associated with anaemia were observed in the high and mid-dose groups at the earlier analysis times. All haematological effects had resolved by the study termination, thus indicating reversibility of the effect. There were no treatment related changes in incidence or types of neoplastic change observed.

As part of the combined dietary chronic toxicity and carcinogenicity study, additional four satellite groups of ten animals of each sex (dose and control groups) were included for evaluation of toxicity and non-neoplastic pathology at 6 months. The results obtained in the first twelve weeks (haematology, biochemistry, urinalysis) were reported separately in a 90 day interim report (Huntingdon Research Centre, 1977). In general, the protocol was similar to the OECD guideline No. 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

Though the disodium etidronate content of the diet was identical (0-10.000 ppm), the

doses in mg/kg bodyweight/day quoted in the 90 day interim report are higher than those in the chronic study report. (top dose of 817 mg/kg in males and 1000 mg/kg in females c.f. 384 and 493 mg/kg for the 2 year study). This can be explained by the higher feed intake as a function of bodyweight. The doses corresponded to 0, 41, 169, 817 mg/kg bw/d for males and 0, 50, 195 and 1000 mg/kg bw/d for females.

A decrease in red blood cell parameters was seen in the top dose group for both sexes, and for males at 2000 ppm by week 12. There was evidence of prolonged anaemia in both sexes at 10000 ppm. Severe pallor of the skin of the top dose group animals and slight pallor in rats receiving 2000 ppm was seen. These observations are consistent with perturbation of iron homeostasis.

The effects are consistent with the chelating properties HEDP and its salts causing a reduction in bioavailability of iron. In principle, two NOAEL values – one for adult (78 mg/kg bw/d), one for juvenile animals during their growth phase (41 mg/kg bw/d) – can be derived on the basis of the results (anaemia) of the two key studies. A NOAEL of 41 mg/kg bw/d already takes into account the most susceptible subgroup (adolescent animals).

The NOAEL value can then be adjusted for the molecular weight of the tetrasodium salt resulting in 48 mg/kg/day.

INHALATION ROUTE:

Acute and short-term inhalation studies of questionable reliability in rats indicate local irritating effects in the nasopharyngeal zone after exposure to aerosols of unknown particle range containing unknown compositions of HEDP and its salts. The pH values were not stated in the reports. As an example, the pH values of aqueous solutions at around 1% can vary between < 2 for the parent polyprotic etidronic acid and 11-12 for the tetrasodium etidronate salt (see pKa values in the chapter “dissociation constant”). Acidic or basic pH conditions significantly determine the induction of local effects and thus could explain the observed irritation.

Justification for grouping:

See CSR Annex I or IUCLID section 13.

Justification for classification or non-classification

Based on the available data, adverse effects on iron homeostasis were not considered to be severe enough to trigger classification. Therefore a classification for STOT-RE is not proposed for sodium salts of HEDP.