Registration Dossier

Administrative data

Description of key information

There are no data for HEDP potassium salts. Therefore, data were read-across from HEDP sodium salts.

A NOAEL for HEDP-2Na of 41 mg/kg bw/d was derived based on anaemia 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

Referenceopen allclose all

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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Conducted prior to guideline adoption.
GLP compliance:
no
Remarks:
Study was conducted prior to GLP.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.


Key result
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; equivalent to 34 mg/kg bw/day active acid
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; equivalent to 139 mg/kg bw/day active acid
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:
chronic toxicity: oral
Type of information:
experimental study
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:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
Conducted prior to adoption of OECD guideline
Deviations:
yes
Remarks:
Uses only 40 animals
GLP compliance:
no
Remarks:
Study was conducted prior to GLP.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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:
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:
main: 104 w
satellite: 26 w
Frequency of treatment:
continuous
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:
Main = 40
Satellite = 10
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.


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
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:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
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:
no effects observed
Details on results:
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:
78 mg/kg bw/day (nominal)
Basis for effect level:
other: adult animals (2000 ppm)
Remarks on result:
other: equivalent to 64 mg/kg bw/day active acid
Critical effects observed:
not specified
Conclusions:
In a well conducted and reported, pre-GLP, dietary Combined Chronic Toxicity / Carcinogenicity study (reliability score 2), conducted using a protocol similar to OECD 453, the NOAEL for Complexing Agent - Henkel (sodium salt of 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate)) was 78 and 96 mg/kg bw/day for males and females, respectively, based on anaemia at higher doses.

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. 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 453 guideline.

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. An additional 4 satellite groups of 10 animals of each sex (dose and control groups) are included for evaluation of toxicity and non-neoplastic pathology at 6 months. 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.

In particular there were no treatment related changes in incidence or types of neoplastic change observed.

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. Haematological disturbances associated with anaemia were observed in the high and mid-dose groups. However, all haematological effects were only seen at the earlier analysis times and had resolved by the study termination, thus indicating reversibility of the effect. The anaemia effects in the early phase of the study are described in more detail in the second entry in this chapter. 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 effects are consistent with the chelating properties of the test substance and probable reduction in bioavailability of iron. A NOAEL of 2000 ppm (78 mg/kg) was assigned for adult animals on the basis of anaemia at the higher doses. The investigations conducted on the satellite groups are reported separately and lead to a different NOAEL of 500 ppm for juvenile animals.

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)

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:

Two key studies are available for repeated dose toxicity by the oral route (dietary) in rats, a 90 day (OECD TG 408) (HRC, 1977) and a combined chronic toxicity and carcinogenicity study (OECD 453) (HRC, 1979). 

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 HEDP-2Na. 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, haematology, 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, an 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 TG 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 of 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 active acid resulting in 34 mg active acid/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 read-across data for HEDP sodium salts, 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 potasium salts of HEDP according to Regulation (EC) No 1272/2008.