(1-hydroxyethylidene)bisphosphonic acid, potassium salt

Carcinogenicity

Description of key information

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

In a well reported dietary Combined Chronic Toxicity / Carcinogenicity study (reliability score 2), conducted using a protocol similar to OECD Test Guideline 453 and pre-GLP, no evidence of neoplastic activity for HEDP (2 -3Na) tested up to 384 and 493 mg/kg bw/day for males and females respectively was observed (Huntingdon Research Centre, 1979).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting 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)

Principles of method if other than guideline:

Method: other

GLP compliance:

no

Remarks:

pre-GLP

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: 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 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:

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 weeks

Frequency of treatment:

continuous

Post exposure period:

None

Dose / conc.:

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

Remarks:

approximately 500 ppm for males

Dose / conc.:

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

Remarks:

approximately 2000 ppm for males

Dose / conc.:

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

Remarks:

approximately 10000 ppm for males

Dose / conc.:

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

Remarks:

approximately 500 ppm for females

Dose / conc.:

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

Remarks:

approximately 2000 ppm for females

Dose / conc.:

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

Remarks:

approximately 10000 ppm for females

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: 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: 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: Yes
- 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: Yes
- 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 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, 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

Description (incidence and severity):

At 10 000 ppm, severe pallor of skin was observed from week 6, however, regressed from week 35 and was back to normal by week 68. At 2000 ppm, 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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Survivors at study termination: Males: 14 (control), 16 (500 ppm), 23 (2000 ppm) and 20 (10 000 ppm) ; Females: 14 (controls), 22 (500 ppm), 19 (2000 ppm) and 19 (10 000 ppm).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 10 000 ppm group, lower bodyweight gain in males was observed during the first 13 weeks only and in females during the first 12 weeks and from weeks 26-52. However, no consistent intergroup differences occurred overall.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No consistent intergroup differences occurred indicating treatment-related changes. However, doses received in the main study were considerably lower than those in the 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:

no effects observed

Description (incidence and severity):

No consistent treatment-related differences were observed between treated and control animals.

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

effects observed, non-treatment-related

Description (incidence and severity):

Slight differences were noted which were attributed to slight differences in food consumption between groups.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Perturbations were recorded at early times although no treatment-related effects that persisted until 104 weeks were observed. In the animals treated with 10 000 ppm, lower values relating to red cell parameters in both sexes were observed during weeks 5, 7 and 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 was observed at week 25 and 26. At week 5, all animals in the 10 000 ppm dose group showed slight polychromasia and/or hypochromasia. These observations were extended to the lower group at week 7 and showed lower red cell values in the 2000 ppm and 10 000 ppm dose group males. All rats treated with 10 000 ppm had several abnormalities of the red blood cells. Examination of blood films from males in the 2000 ppm dose group revealed only slight to moderate anisocytosis, polychromasia and hypochromasis in some of the rats. At week 12, ana emia was still observed among males and females receiving 10 000 ppm and among males only in the 2000 ppm dose group. Several blood cell abnormalities were observed in the 10 000 ppm male dose group. By 25 weeks, the values relating to red cell parameters were similar to controls for the 2000 ppm dose group and the packed cell volume and haemoglobin concentration were only marginally lower in the 10 000 ppm dose group. However, the red cell count for 10 000 ppm group was higher than in the control dose group. An increase in microcytosis and presence of giant platelets were observed in some of the 10 000 ppm treated animals. Red cell fragility examinations conducted at week 26 indicated the red cells from animals treated with 10 000 ppm could shrink in the presence of normal plasma. It was considered that the microcytosis observed in association with the anaemia in rats in the 10 000 ppm dose group 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 10 000 ppm dose group occurred during weeks 6 and 7 as well as in the 2000 ppm male dose group when the observations were extended to the lower group at week 7. Although marginally higher, lymphocyte count was observed in the 500 ppm and 2000 ppm female dose groups during week 7 although not statistically significant. No differences were observed at later sampling times. There was a marginally higher platelet count in the high dose male group during weeks 12 and 25.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 10 000 ppm, higher serum alkaline phosphatase in males at week 5, 7 and 12 was reported. No effects at lower doses were observed. Although some inter-group differences occurred, these were not considered as consistent, outside of normal ranges or was considered of not being of toxicological relevance.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Marginally increased urine volume was found in males in all the dose groups at week 7, although 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.

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):

In the 10 000 ppm dose group, lower liver weights in both sexes as well as lower kidney weights in males at 26 weeks, although not at 104 weeks, were reported. In the 2000 ppm dose group, lower liver weights were recorded for males at 26 weeks, although not at 104 weeks. In the 500 ppm dose group, lower liver weights among females at 26 weeks, although not 104 weeks, were reported.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were observed in any dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the 2000 ppm and 10 000 ppm male dose groups and in the 10 000 ppm female dose group at week 26 but not week 104, treatment-related changes in the spleen, particularly, a lack of iron were observed. No evidence of treatment-related effects was found in the 500 ppm dose 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.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No increased incidence of neoplastic lesions was observed in treated groups at 104 weeks.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 493 mg/kg bw/day

Sex:

female

Basis for effect level:

other: No carcinogenic effect at any dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

>= 384 mg/kg bw/day

Sex:

male

Basis for effect level:

other: No carcinogenic effects at any dose tested

Key result

Critical effects observed:

not specified

The tumour types and incidence were normal for this strain. The following gives frequency (%) of neoplastic lesions of different types for control, 500 ppm, 2000 ppm, 10 000 ppm 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:

In a well reported dietary Combined Chronic Toxicity / Carcinogenicity study (reliability score 2), conducted using a protocol similar to OECD Test Guideline 453 and pre-GLP, no evidence of neoplastic activity for HEDP (2-3Na) tested up to 384 and 493 mg/kg bw/day for males and females respectively was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

384 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Klimisch score 2

System:

other: No carcinogenic effect at any dose tested

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available carcinogenicity study, HEDP-xK does not require classification for carcinogenicity according to Regulation (EC) No 1272/2008.

Additional information

In a well reported dietary Combined Chronic Toxicity / Carcinogenicity study (reliability score 2), conducted using a protocol similar to OECD Test Guideline 453 and pre-GLP, HEDP (2-3Na) was administered to Sprague-Dawley rats (40/sex/dose) in the diet for 104 weeks. The concentrations tested included 500, 2000 or 10 000 ppm which is equivalent to 19, 78 or 384 mg/kg/day for males and 24, 96 or 493 mg/kg/day for females. 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, as well as gross and microscopic examinations. No evidence of neoplastic activity was observed at any dose groups. Perturbations of haematological parameters were observed in the highest dose groups (Huntingdon Research Centre, 1979).