Registration Dossier

Diss Factsheets

Administrative data

Description of key information

In the key 90-day oral (feeding) repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for DTPMP (5-7Na) 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 test material contained 46.9% w/w active acid; according to the study report the administered test substance was corrected for purity) (Central Toxicology Laboratory, 1998). 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 (5-7Na) on calcium homeostasis, however, without causing any changes in calcium plasma levels.


The effects observed in this study and the resulting NOAEL will be re-evaluated on completion of the ongoing OECD 443 test. 


 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
Species:
rat
Strain:
other: Alpk APfSD (Wistar derived)
Details on species / strain selection:
The Alpk APfSD strain of rat was used because of the substantial background data available for this strain, in this Laboratory, relating to studies of this type.
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): 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
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 Dequest 2066A 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 Dequest 2066A. The homogeneity of Dequest 2066A in CTL diet was determined by analysing samples from the high dose level. The chemical stability of Dequest 2066A 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
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
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.
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 10000 ppm group was slightly below control values during weeks 7-13. In this group food consumption for one cage of females was also consistently lower than control values, whereas the food consumption for the other 2 cages in this group was lower than the controls only in isolated weeks. These small decreases in food consumption are considered not to be 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 haemaglobin 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):
No effects.
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.
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
Critical effects observed:
yes
Lowest effective dose / conc.:
841.9 mg/kg bw/day (actual dose received)
System:
cardiovascular
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

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



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 (5-7Na) 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 (5-7Na) on calcium homeostasis, however, without causing any changes in calcium plasma levels.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
82.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
cardiovascular
Organ:
blood

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

There were no repeated dose toxicity data for DTPMP (1-3Na), therefore data were read-across from the Category member DTPMP (5-7Na).


See attachment to Section 13 for justification of read-across.


 


In the key 90-day oral (feeding) repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP, groups of 12 male and 12 female Wistar-derived rats were fed diets containing 0 (control), 100, 1000 or 10000 ppm DTPMP (5-7Na) (equivalent to 8.2, 82.5 and 841.9 mg active acid/kg bw/day in males and 9.2, 92.3 and 902.6 mg active acid/kg for females; the test material contained 46.9% w/w active acid; according to the study report the administered test substance was corrected for purity) for 90 consecutive days. Clinical observations were made daily, bodyweights and food consumption were measured on a regular basis. Ophthalmoscopic and haematological examinations, clinical chemistry and urinalysis were conducted. At the end of the exposure period all animals were killed and a full microscopic examination was conducted. Bone mineral density was evaluated for bone collected at termination.


There were no deaths and the majority of parameters were unaffected by the treatment. Minor changes in certain haematological parameters, with a statistically significant increase in red blood cell count resulting in a statistically significant decrease in mean cell volume and mean cell haemoglobin concentration, were noted at the highest dose. Total serum iron was decreased in high dose females only, while total serum iron binding capacity was increased in high dose males only. A reduction in iron complexes and reduced pigmentation for age was noted in the spleens in high dose animals of both sexes. The changes in haematological parameters and serum iron and binding capacity were considered by the study authors to be perturbations of iron homeostasis as a result of the iron binding capacity of this salt, which is a chelating agent. No effects on the density of cortical bone were seen. The total bone density and that of trabecular bone was increased in both sexes at the top dose but no effects were seen at other doses. The incidence of microlithiasis (formation of minute calculi) in kidneys of females was reduced at all dose levels. These changes on bone density and the reduced level of microlithiasis in the kidneys were considered to be indicative of an effect on calcium homeostasis due to the chelating characteristics of the test substance, which occurred without any concurrent alteration of calcium plasma levels. According to the study report, the changes were not considered to be of toxicological significance, and the NOAEL was concluded to be 10000 ppm. However, as a precautionary approach and in view of the haematological disturbances and the effects on the bone density in the top dose group, the NOAEL used for risk assessment was concluded to be 82.5 mg DTPMP active acid/kg bw/day (1000 ppm). The effects observed in this study and the resulting NOAEL will be re-evaluated on completion of the ongoing OECD 443 test. 


 


In a supporting 90-day oral (feed) repeated dose toxicity study (SafePharm Laboratories, 1982d), not conducted according to OECD Test Guidelines and partially in compliance with GLP, DTPMP (5-7Na) was administered at 6000 ppm (calculated as approximately equivalent to 151 and 195 mg active acid/kg bw/day, in males and females, respectively) continuously in diet, to five male and five female Sprague-Dawley rats for 90 days. There were no adverse effects, including no mortality, clinical effects, food consumption or body weight changes, or effects on haematology and clinical chemistry parameters. No urinalysis was conducted. At the end of the exposure period the animals were sacrificed and a gross macroscopic examination was conducted. No abnormal findings were observed at necropsy. No histopathological examination was conducted. The NOAEL was concluded to be ≥6000 ppm (equivalent to 151 and 195 mg active acid/kg bw/day, in males and females, respectively).

Justification for classification or non-classification

Based on the available data for DTPMP (5-7Na), no classification for repeated dose toxicity is required for DTPMP (1-3Na) according to Regulation (EC) No. 1272/2008. Although the NOAEL for effects on haematological parameters was within the classifiable range, the effects are not sufficiently severe to trigger classification.