Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies). Study conducted prior to adoption of OECD guideline.
Deviations:
yes
Remarks:
Only 40 animals used (50 in guideline)
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

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

Administration / exposure

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
Details on study schedule:
none
Doses / concentrationsopen allclose all
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:
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

Examinations

Parental animals: Observations and examinations:
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
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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 384 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: only as far as effects on reproductive organs are considered
Remarks on result:
other: equivalent to 316 mg/kg bw/day active acid
Key result
Dose descriptor:
NOAEL
Effect level:
>= 493 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: only as far as effects on reproductive organs are considered
Remarks on result:
other: equivalent to 406 mg/kg bw/day active acid

Results: F1 generation

Effect levels (F1)

Key result
Remarks on result:
other: F0 were not mated

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

 

 

Group

No. of rats with:

1♂

2♂

3♂

4♂

1♀

2♀

3♀

4♀

 

D

T

D

T

D

T

D

T

D

T

D

T

D

T

D

T

REPRODUCTIVE SYSTEM

Testes

Arrest spermatogenesis / atrophy / dilation of tubules / peri-arteritis

7

2

12

2

8

6

4

6

 

 

 

 

 

 

 

 

Dystrophic mineralisation of blood vessels

 

1

 

 

3

1

1

 

Miscellaneous

including congestion, haemorrhage, oedema, inflammation in the epididymis, spermatocele in the epididymis

 

 

 

1

2

 

1

 

 

 

 

 

 

 

 

 

Prostate

Prostatis

3

1

2

 

 

 

 

 

 

 

 

 

 

 

 

 

Low epithelium / hyperplasia of epithelium

2

 

 

Seminal vesicles

Lack of secretion within acini

3

2

 

 

 

1

 

 

 

 

 

 

 

 

 

 

Distended acini / low epithelium

 

 

1

 

 

 

 

1

Inflammatory cell infiltration / abscess

 

1

2

 

2

 

 

 

 

D = Rats dying or killed during the study                      T = Rats killed at termination of study

  

 

 

Group

No. of rats with:

1♂

2♂

3♂

4♂

1♀

2♀

3♀

4♀

 

D

T

D

T

D

T

D

T

D

T

D

T

D

T

D

T

Ovaries

Lack of atretic / cystic follicles / cysts

 

 

 

 

 

 

 

 

3

1

1

4

3

1

2

5

Lack of corpora lutea

1

 

 

1

 

7

1

5

Corpora lutea +/- haemorrhage

 

2

 

 

 

 

1

 

Miscellaneous

including pigment deposition cyst in oviduct

 

 

 

 

 

 

 

 

 

1

 

 

1

1

 

 

Uterus

Congestion of blood vessels / mononuclear cell aggregations

 

 

 

 

 

 

 

 

1

 

 

1

 

2

 

 

Dilation lumen / endometrial gland

 

1

1

3

1

1

Hyperplasia / squamous metaplasia

 

 

 

1

 

1

Miscellaneous

including intussusception infarction, necrosis in lumen, polyp

 

 

 

 

 

 

 

 

1

 

 

1

 

1

1

1

Mammary gland

Hyperplasia / galactocele / dilated duct +/- increased connective tissue / fibrosis

 

 

4

1

3

2

0

 

8

7

2

12

4

9

3

13

Miscellaneous

including pigmented macrophages inlammatory cell infiltration

 

 

 

 

 

 

1

 

 

 

 

 

 

 

1

 

 

D = Rats dying or killed during the study                      T = Rats killed at termination of study

Applicant's summary and conclusion

Conclusions:
Macroscopic and microscopic examination did not reveal any abnormalities in gonads (ovaries & testes) and accessory reproductive organs (uterus, cervix, mammary, endometrial & mammary glands, testes, preputial gland, prostate, & seminal vesicles).
The NOAEL for effects on reproductive organs in this study is therefore >= 384 mg/kg/day in male rats and >= 493 mg/kg/day in female rats.
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). The animals 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).

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.

The NOAEL for effects on reproductive organs in this study is therefore >= 384 mg/kg/day in male rats and >= 493 mg/kg/day in female rats.