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Description of key information

In a good quality 90-day feeding study (CTL, 1998) conducted to OECD 408 and GLP, groups of 12 male and 12 female Wistar-derived rats were fed diets containing 0 (control), 100, 1000 or 10000 ppm (equivalent to 8.2, 82.5 and 841.9 mg/kg bw/day in males and 9.2, 92.3 and 902.6 mg/kg for females) test material (sodium salt of DTPMP, xNa) for 90 consecutive days. There were no deaths and the majority of parameters were unaffected by the treatment. Minor changes in certain 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. 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 the test material on calcium homeostasis, however, without causing any changes in calcium plasma levels. Therefore, the changes were not considered to be of toxicological significance, and the NOAEL was 10000 ppm (equivalent to to 841.9 and 902.6 mg/kg bw/day of the salt in males and female, respectively). However, it is the opinion of the author of this study summary that anaemia in humans is of concern, and therefore the NOAEL is reduced to 1000 ppm (equivalent to 82.5 and 92.3 mg active acid/kg bw/day for males and females, respectively). The test material contained 46.9% active acid. According to the study report the administered test substance had been corrected for purity.

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Alpk APfSD (Wistar derived)
Sex:
male/female
Details on test animals 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): 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 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 bymixing 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; equvalent to 100 ppm nominal in diet; calculated intakes from food consumption data
Dose / conc.:
9.2 mg/kg bw/day (nominal)
Remarks:
Females; equvalent 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:
12
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)
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
Mortality:
no mortality observed
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):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no deaths or treatment-related clinical signs of toxicity.

BODY WEIGHT AND WEIGHT GAIN: 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): 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 treatment-related effect.


OPHTHALMOSCOPIC EXAMINATION: No effects on the appearance of the eyes.

HAEMATOLOGY: 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 Dequest 2066A, which is a chelating agent. All of these observations are considered to be without toxicological significance.

CLINICAL CHEMISTRY: 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 postassium levels were statistically significantly increased in females at 10000 ppm. These changes were small in magnitude and not considered toxicologically relevant. 

URINALYSIS: No effects.

ORGAN WEIGHTS: 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 PATHOLOGY: No findings.

HISTOPATHOLOGY: 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.

OTHER: 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:
test mat.
Sex:
male
Basis for effect level:
other: Expressed as active acid in males
Key result
Dose descriptor:
NOAEL
Effect level:
92.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Expressed as active acid in females
Critical effects observed:
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 a good quality 90-day feeding study (reliability score 1) conducted to OECD 408 and GLP, the NOAEL for Dequest 2066A was 1000 ppm (equivalent to 82.5 and 92.3 mg/kg bw/day of active acid in males and females, respectively).
Executive summary:

In a good quality 90-day feeding study (reliability score 1) conducted to OECD 408 and GLP, groups of 12 male and 12 female Wistar-derived rats were fed diets containing 0 (control), 100, 1000 or 10000 ppm (equivalent to 8.2, 82.5 and 841.9 mg/kg bw/day in males and 9.2, 92.3 and 902.6 mg/kg for females) Dequest 2066A for 90 consecutive days. Clinical observations, bodyweights and food consumption were measured. Ophthalmoscopic and haematology examinations, clinical chemistry and urinalysis were conducted. At the end of the exposure period all animals were killed and a full microscopic examination 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 (red blood cell count was significantly increased, mean cell volume and mean cell haemoglobin concentration were significantly decreased) were noted at the highest dose. 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 Dequest 2066A on calcium homeostasis, however, without causing any changes in calcium plasma levels. Therefore, the changes were not considered to be of toxicological significance, and the NOAEL was 1000 ppm (equivalent to to 82.5 and 92.3 mg/kg bw/day of active acid in males and females, respectively).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
82.5 mg/kg bw/day

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

The key study was the most reliable study available for the sodium salt of DTPMP.

The use of read-across data between members of the category is in accordance with the rationale outlined in the Toxicological information endpoint summary of the IUCLID and Section 1.4 of the CSR.

Discussion of trends in the DTPMP category

In a good quality 90-day feeding study (CTL, 1998) conducted to OECD 408 and GLP, groups of 12 male and 12 female Wistar-derived rats were fed diets containing 0 (control), 100, 1000 or 10000 ppm (equivalent to 8.2, 82.5 and 841.9 mg/kg bw/day in males and 9.2, 92.3 and 902.6 mg/kg for females) DTPMP-xNa for 90 consecutive days. Clinical observations, bodyweights and food consumption were measured. Ophthalmoscopic and haematology examinations, clinical chemistry and urinalysis were conducted. At the end of the exposure period all animals were killed and a full microscopic examination 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. In view of the haematological disturbances and the effects on the bone density in the top dose group, the NOAEL was considered to be 82.5 mg DTPMP salt/kg bw/d (1000 ppm). The test material contained 46.9% active acid. According to the study report the administered test substance had been corrected for purity.

In a supporting limited 90-day feeding study (Safepharm, 1982) in which Briquest 543-33S was administered at 6000 ppm (calculated by previous reviewer as approximately equivalent to 151 and 195 mg/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 animals were killed and a gross macroscopic examination conducted. No abnormal findings were observed. No histopathological examination was conducted. The NOAEL was concluded to be ≥6000 ppm (equivalent to 151 and 195 mg/kg bw/day, in males and females, respectively).



Justification for classification or non-classification

Based on the available data for DTPMP sodium salts, no classification for repeated dose toxicity is required for DTPMP (5-7Na) 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.