Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05.08.1997 to 22.04.1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate
EC Number:
701-216-4
Molecular formula:
DTPMP-5Na C9H23N3Na5O15P5 DTPMP-6Na C9H22N3Na6O15P5 DTPMP-7Na C9H21N3Na7O15P5
IUPAC Name:
octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Alpk APfSD (Wistar derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zeneca Pharmaceuticals
- Age at study initiation: 28 days
- Weight at study initiation: 159g (males); 133g (females)
- Fasting period before study: No
- Housing: Four per cage in multiple rat racks.
- Diet (e.g. ad libitum): Control CT1 diet until the start of the study and the appropriate experimental diet following this, ad libitum
- Water (e.g. ad libitum): Mains tap water, ad libitum
- Acclimation period: One week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 05.08.1997 To: 22.04.1998

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route via the diet was chosen for administration of the test substance as this represents a possible route of human exposure
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: No data


DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were made in 30 kg batches from premixes prepared by mixing the appropriate amount of the test substance with up to 7 lots of 1 kg batches of milled diet. The premixes were then added to the appropriate amount of diet and mixed thoroughly.
- Mixing appropriate amounts with (Type of food): Milled CTL diet (no further information)
- Storage temperature of food:

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of diet from the control and 1000 ppm groups were analysed pre-study and once during the study for achieved concentration of the test substance. The homogeneity of the test substance in CTL diet was determined by analysing samples from the high dose level. The chemical stability of the test substance in diet was determined at this dose level over a period of up to 36 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuous
Doses / concentrationsopen allclose all
Dose / conc.:
8.2 mg/kg bw/day (nominal)
Remarks:
Males; equivalent to 100 ppm nominal in diet; calculated intakes from food consumption data
Dose / conc.:
9.2 mg/kg bw/day (nominal)
Remarks:
Females; equivalent to 100 ppm nominal in diet; Calculated intakes from food consumption data
Dose / conc.:
82.5 mg/kg bw/day (nominal)
Remarks:
Males; equivalent to 1000 ppm nominal in diet; Calculated intakes from food consumption data
Dose / conc.:
92.3 mg/kg bw/day (nominal)
Remarks:
Females: equivalent to 1000 ppm nominal in diet; Calculated intakes from food consumption data
Dose / conc.:
841.9 mg/kg bw/day (nominal)
Remarks:
Males; equivalent to 10000 ppm nominal in diet; Calculated intakes from food consumption data
Dose / conc.:
902.6 mg/kg bw/day (nominal)
Remarks:
Females; equivalent to 10000 ppm nominal in diet; Calculated intakes from food consumption data
No. of animals per sex per dose:
12M 12F
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected by the Sponsor based on previous toxicity data.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post-exposure recovery period in any group.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included: significant changes in clinical condition or behaviour.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the same time as body weight measurements.


BODY WEIGHT: Yes
- Time schedule for examinations: immediately before start of treatment and then on the same day of each week until termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Recorded continuously throughout the study for each cage of rats.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Food utilisation value per cage was calculated as the bodyweight gained by the rats in the cage per 100 g of food eaten.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all examined pre-treatment, and those of high dose and control groups during the week prior to termination.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All surviving animals.
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination.
- Animals fasted: No
- How many animals: All surviving animals.
- Parameters checked in table [No.1] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: In the week prior to termination over a period of 16-18 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during collection.
- Parameters checked in table [No.1] were examined.


NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Other examinations:
Bone density evaluation - The left femur from each animal was thawed for one hour at room temperature prior to analysis of bone density. Imaging of bone density was carried out using an "XCT960A" pQCT bone densitometer using a second generation rotate-translate computed tomographic technique. A scout scan of the femoral condolytes was obtained to identify the standard reference line to allow accurate positioning of the bone. Multiple slice traverse image sets were obtained from the distal femur to draw regions of interest on the slice images, and bone mineral density was calculated via COMAC European Forearm hydroxyapatite phantom. A total bone mineral density measurement was also obtained.
Statistics:
Body weights: analysis of covariance (males and females separately).
Food consumption and food utilisation: analysis of variance (males and females separately).
Haematology, clinical chemistry and urine analysis: analysis of variance (male and female data analysed together).
Organ weights: analysis of variance.
Least squares mean for each group were calculated. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least-squares mean. Each treatment group least-squares mean was compared with the control group least-squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths or treatment-related clinical signs of toxicity.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The group mean bodyweights for the male rats in the 1000 ppm group diverged slightly from controls in the first few weeks of the study. The differences were not statistically significant, and were mainly due to a reduction in one animal. Overall, there was no treatment-related effect on body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The group mean food consumption for males in the 1000 ppm group was slightly below control values during weeks 7-13. The difference was mainly due to one female, and was not considered of toxicological significance.
Food efficiency:
no effects observed
Description (incidence and severity):
No treatment-related effect.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects on the appearance of the eyes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in red blood cell levels in males and females at 10000 ppm. Mean cell volume and mean cell haemoglobin were also decreased at this dose.  Haemoglobin and mean cell haemoglobin concentration were significantly decreased in females at top dose. Total iron binding capacity in the serum of males only were increased and the total serum iron decreased in females only.  All changes described were statistically significant. Perls' staining for iron complexes showed decreases in the spleens of both sexes.  Thus, the findings noted in these haematological parameters and serum iron and binding capacity are likely to result from a perturbation of iron homeostasis, which is supported by the reduction of staining in the spleen. The effects are therefore due to the iron binding characteristics of DTPMP (5-7Na), which is a chelating agent. All of these observations are considered to be without toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Plasma albumin was statistically significantly increased in males at 1000 ppm.  This increase was not dose-related and therefore considered treatment-related. Plasma creatine kinase activity and potassium levels were statistically significantly increased in females at 10000 ppm. These changes were small in magnitude and not considered toxicologically relevant.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in plasma parathyroid hormone levels in either sex in any dose group compared with the control values.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment related findings in any dose group in either sex or any urine parameters evaluated.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was a small but statistically significant decrease in the group mean liver weight (absolute) of male rats in the 10000 ppm group. The effect was not considered of toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Reduced pigmentation for age in spleens of male and female rats in the 10000 ppm group. Perl' staining indicated marked reduction in positive staining (for iron complexes) for age in males and a marked  or slight reduction in positive staining for age in females receiving 10000ppm. There was a reduced incidence of microlithiasis in kidneys of females at all dose levels. These findings were considered not to be of toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No effect on density of cortical bone. The total bone density and that of trabecular bone was increased in both females and males at highest dose.  No effects seen at other doses.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
82.5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Expressed as active acid in males
Sex:
male
Basis for effect level:
haematology
other: significantly increased bone density in both sexes at 10000 ppm
Key result
Dose descriptor:
NOAEL
Effect level:
92.3 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
Expressed as active acid in females
Sex:
female
Basis for effect level:
haematology
other: significantly increased bone density in both sexes at 10000 ppm

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 3 Selected haematology and clinical chemistry

Dietary concentrations (ppm)

0

100

1000

1000

0

100

1000

10000

male

female

Number of animals/group

12

12

12

12

12

12

12

12

Haematology

 

 

 

 

 

 

 

 

Haemoglobin (g/dl)

 14.9

 15.0

 14.8

 14.5

 14.9

15.1 

14.9 

14.2* 

Haematocrit

 0.465

 0.467

0.462 

0.459 

 0.452

0.457 

0.452 

0.438 

Red blood cell count (x10**12/l)

 8.75

8.77 

8.75 

9.49** 

 8.22

8.34 

8.13 

8.65* 

Mean cell volume (fl)

 53.1

 53.3

52.8 

49.6** 

 55.1

54.9 

55.7 

50.8** 

Mean cell haemoglobin (pg)

 17.0

17.2 

17.0 

15.4** 

 18.2

18.2 

18.9 

16.5** 

Mean cell haemoglobin concentration (g/dl)

32.0

32.2

32.1

31.6

33.1

33.1

33.0

32.4**

Blood chemistry

 

 

 

 

 

 

 

 

Plasma creatinine kinase (IU/l)

 129.4

141.0 

126.5 

151.1 

 123.1

121.4 

133.1 

155.1* 

Plasma potassium (mmol/l)

 4.80

4.94 

5.18 

5.06 

 5.00

5.13 

5.14 

5.67* 

* P0.05   ** P0.01



Applicant's summary and conclusion

Conclusions:
In the 90-day oral (feeding) repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for DTPMP (5-7 Na) was concluded to be 1000 ppm (equivalent to 82.5 and 92.3 mg/kg bw/day of active acid in males and females, respectively). The NOAEL was based on minor changes in haematological parameters (red blood cell count was significantly increased, mean cell volume and mean cell haemoglobin concentration were significantly decreased) were noted at the highest dose tested. There was also a decreased incidence in Perls' staining of the spleen. Bone density was significantly increased in both sexes in the highest dose group, and the incidence of microlithiasis in the kidney was reduced at all dose levels. These changes are indicative of the influence of DTPMP (5-7 Na) on calcium homeostasis, however, without causing any changes in calcium plasma levels.