Tetrasodium (1-hydroxyethylidene)bisphosphonate

Carcinogenicity

Description of key information

For sodium salts of HEDP, a study comparable to OECD 453 conducted with disodium etidronate showed no indication of carcinogenic potential.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: 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

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

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Study conducted prior to adoption of OECD guideline.

Deviations:

yes

Remarks:

Only 40 animals used (50 in guideline)

GLP compliance:

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 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:

104 w

Frequency of treatment:

continuous

Post exposure period:

None

Dose / conc.:

500 ppm

Remarks:

Males: 19 mg/kg bw/day (corresponding doses expressed in terms of material as supplied)
Females: 24 mg/kg bw/day (orresponding doses expressed in terms of material as supplied)

Dose / conc.:

2 000 ppm

Remarks:

Males: 78 mg/kg bw/day (corresponding doses expressed in terms of material as supplied)
Females: 96 mg/kg bw/day (orresponding doses expressed in terms of material as supplied)

Dose / conc.:

10 000 ppm

Remarks:

Males: 384 mg/kg bw/day (corresponding doses expressed in terms of material as supplied)
Females: 493 mg/kg bw/day (orresponding doses expressed in terms of material as supplied)

No. of animals per sex per dose:

40

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.

Relevance of carcinogenic effects / potential:

In a study similar to OECD 453, there was no evidence of neoplastic activity for Complexing Agent-Henkel (sodium salt of 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate) tested up to 384 and 493 mg/kg bw/d for males and females.

Dose descriptor:

NOAEL

Effect level:

>= 384 mg/kg bw/day

Sex:

male

Basis for effect level:

other: No carcinogenic effects at any dose tested

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

NOAEL

Effect level:

>= 493 mg/kg bw/day

Sex:

female

Basis for effect level:

other: No carcinogenic effect at any dose tested

Remarks on result:

other: Effect type: carcinogenicity

The tumour types and incidence were normal for this strain. The following gives frequency (%) of neoplastic lesions of different types for control, 500ppm, 2000ppm, 10000ppm respectively (m = males, f = females):

Tissue	Dose group
	Control	500 ppm	2000 ppm	10000 ppm
Cutaneous	14 (m)	25 (m)	35 (m)	30 (m)
Subcutaneous	43 (m) 71 (f)	38 (m) 78 (f)	35 (m) 68 (f)	25 (m) 74 (f)
Hypophyseal	21 (m) 64 (f)	12 (m) 39 (f)	9 (m) 32 (f)	10 (m) 32 (f)
Thyroid	0 (m)	6 (m)	9 (m)	0 (0)
Adrenal	0 (m) 14 (f)	12 (m) 13 (f)	17 (m) 11 (f)	15 (m) 5 (f)
Pancreas	0 (m)	0 (m)	17 (m)	15 (m)
Uterus	0 (f)	4 (f)	16 (f)	11 (f)
Assorted	7 (m) 14 (f)	19 (m) 14 (f)	22 (m) 11 (f)	0 (m) 11 (f)

Conclusions:

not carcinogenic

Executive summary:

In this well-performed study, in which sodium salt of Complexing Agent - Henkel 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate) was administered to Sprague-Dawley-derived rats (40/sex/dose) in the diet for 104 weeks, at dietary concentrations of 500, 2000, 10000 ppm (equivalent to 19, 78 and 384 mg/kg/day for males, and 24, 96, 493 mg/kg/day for females). This 'main' group was primarily used to assess the carcinogenic potential of the test substance. A satellite group (10/sex/dose) received the same doses for 26 weeks, and was used to assess general toxicity (blood and urinary assessments made on this group only). The main groups were observed for mortality, clinical signs of toxicity, food and water consumption, ophthalmoscopy, and gross and microscopic examinations. There was no evidence of neoplastic potential, or other chronic toxicity effects for disodium etidronate apart from the perturbations of haematological parameters that were observed in the highest dose groups (discussed in Section 7.5.1). The NOAEL for carcinogenicity was 384 and 493 mg/kg bw/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Based on the available carcinogenicity study tetrasodium HEDP does not require classification for carcinogenicity.

Additional information

In a well conducted and reported, pre-GLP, dietary Combined Chronic Toxicity / Carcinogenicity study (Huntingdon Research Centre, 1979), conducted using a protocol similar to OECD 453, there was no evidence of neoplastic activity for Complexing Agent - Henkel (sodium salt of 1-hydroxythane-1,1-diphosphonic acid (disodium etidronate)) tested up to 384 and 493 mg/kg bw/day for males and females, respectively.

Justification for grouping:

See CSR Annex I or IUCLID section 13.