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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 August 2010 to 28 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with an internationally recognised method

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-Day Oral Toxicity Study in Rodents
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine
EC Number:
239-590-1
EC Name:
Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine
Cas Number:
15541-60-3
Molecular formula:
C3H6N6.xH4O7P2
IUPAC Name:
diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): MPP
- Appearance: white powder
- Analytical purity: 97.04%
- Lot/batch No.: G10170

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: reputable laboratory animal supplier
- Age at study initiation: 29 - 35 days
- Weight at study initiation: 108 - 135 g
- Fasting period before study:
- Housing: five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood based material was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week.
- Diet: standard rodent diet, ad libitum
- Water: potable water from the public supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): none, continuous supply of filtered fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: To: 18 October to 15 November 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle:
0 mg/ml (control)
3 mg/ml (15 mg/kg/day)
30 mg/ml (150 mg/kg/day)
200 mg/ml (1000 mg/kg/day)

- Amount of vehicle (if gavage): 5 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of MPP in the doses was quantified by high performance liquid chromatography using UV detection.

The analytical procedure used was successfully validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for MPP in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of MPP in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg/day 3mg/l
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day 30 mg/ml
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day 200 mg/ml
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: The doses used in this study (0, 15, 150 and 1000 mg/kg/day) were with reference to a preliminary study (Huntingdon Life Sciences Report Number CVJ0069). The preliminary study showed that MPP was generally well tolerated at doses of 100, 300 or 1000 mg/kg/day; with no clinical signs or post dose observations considered to be related to treatment. Animals treated at 300 or 1000 mg/kg/day had low overall bodyweight gain and low food consumption and animals at 1000 mg/kg/day had high water consumption and some had pale areas on the kidneys. It was therefore considered that a high dose of 1000 mg/kg/day would be appropriate for the present study, with low and intermediate doses at 15 and 150 mg/kg/day, respectively.

- Rationale for animal assignment: random

- Rationale for selecting satellite groups: no satellite groups used


- Post-exposure recovery period in satellite groups:


- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: one week before treatment then each week throughout the treatment period

FOOD CONSUMPTION: Yes
- Time schedule for examinations: one week before treatment then each week throughout the treatment period


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necroscopy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Haematocrit, Haemoglobin concentration, Erythrocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Total leucocyte count, Differential leucocyte count, Platelet count, Morphological changes, Prothrombin time, Activated partial thromboplastin time.

BLOOD CHEMISTRY: Yes
- Time schedule for collection of blood: at necroscopy
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Total bile acids, Urea, Creatinine, Glucose, Total cholesterol, Sodium, Potassium, Total protein, Albumin


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4, prior to dosing
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy

HISTOPATHOLOGY: Yes
Adrenals, Brain, Femur with joint, Heart, Kidneys, Liver, Lungs, Seminal vesicle, Spinal cord, Sternum, Stomach, Thyroid, Uterus
Other examinations:
Organ weights: Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Uterus with cervix, Ovaries
Statistics:
Grip strength, motor activity, bodyweight, haematology, blood chemistry and organ weight data:
Bartlett's test for variance homogeneity (Bartlett 1937)
Williams’ test (Williams 1971, 1972)
Dunnett's test (Dunnett 1955, 1964)
Shirley's test (Shirley 1977)
Steel's test (Steel 1959)
Fisher’s Exact tests (Fisher 1973)
Angervall and Carlstrom, 1963

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
low in males and females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
low in males
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
high in males and females
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high urea and creatinine concentrations, marginally high total protein concentration and high alanine aminotransferase activity in both sexes.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
high kidney weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
change in appearance of kidneys
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
effects seen in kidneys in both males and females
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths and detailed physical and arena observations revealed no signs that could be attributed to treatment.
There were no toxicologically significant signs after dose administration.


BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain in animals receiving 1000 mg/kg/day was comparable to Control during the first week of treatment. From the second week of treatment to the end of the study, weight gain was markedly low (males 0.57; females 0.73X Control). Overall weight gain in these animals was low (males 0.70; females 0.78X), when compared with Control.

Bodyweight gain at 15 or 150 mg/kg/day was not affected by treatment.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Overall food consumption was low for males treated at 1000 mg/kg/day (0.86X Control); food consumption in females was lower than Control during three out of four weeks of treatment; however overall food consumption for these animals (0.92X) was comparable to Control.

Food consumption in both sexes at 15 or 150 mg/kg/day was not affected by treatment, when compared with Control.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION:
Measured water consumption during Week 4 revealed slightly high values in males receiving 150 mg/kg/day (1.23X Control) and markedly high in animals receiving 1000 mg/kg/day (males 1.87; females 1.88X Control).

Daily visual water consumption appeared high on some days in both sexes receiving 150 mg/kg/day and appeared high throughout the study period in animals treated at 1000 mg/kg/day.

When compared with Control, measured water consumption during Week 4 in both sexes at 15 mg/kg/day and females receiving 150 mg/kg/day was not affected by treatment.

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
Haematological parameters were not affected by treatment.

All differences from Control were present in a single sex, were not statistically significant or considered not to be biologically or toxicologically significant or adverse.

BLOOD CHEMISTRY
Animals receiving 1000 mg/kg/day had markedly high concentrations of urea (males 5.59; females 4.99X Control) and creatinine (males 5.36; females 3.92X Control), total protein concentration was marginally high (males 1.07; females 1.08X Control). Alanine aminotransferase activity was significantly high in males and females (1.46 or 1.64X Control respectively).

Cholesterol concentration was high in both sexes (males 1.21; females 1.14X Control) with statistical significance in males. Aspartate aminotransferase activity was high in males only (1.25X Control). The ratio of albumin to globulin was low in females (0.86X Control). Bile acid concentration appeared low in males; however this may have been the result of high individual variation in the Control. It is considered that these changes were not toxicologically important, as they were either not statistically significant in both sexes, were evident in a single sex or were considered only marginally different from Control.

Electrolyte concentrations were considered not to be affected by treatment.

Blood chemical parameters were not affected by treatment at 15 or 150 mg/kg/day.


URINALYSIS
Not examined


NEUROBEHAVIOUR
Sensory reactivity and grip strength - Sensory reactivity findings showed considerable inter-group variation, with higher than expected numbers of animals showing weak or absent responses. There was, however, no indication of an association with treatment as performance of treated animals was generally similar to that of Control or, where performance was inferior, this was seen in animals at 15 or 150 mg/kg/day. Forelimb and hindlimb grip strength values were unaffected.

Motor activity - Motor activity scores were considered to be unaffected by treatment.

ORGAN WEIGHTS
Bodyweight adjusted mean kidney weight was high in males and females receiving 1000 mg/kg/day (1.45 and 1.86X Control, respectively).

Organ weights in animals receiving 15 or 150 mg/kg/day were not affected by treatment.

GROSS PATHOLOGY
The kidneys of some males and females treated with 1000 mg/kg/day of MPP were enlarged, granular, pale or showed pale areas.


HISTOPATHOLOGY: NON-NEOPLASTIC
The kidneys of males treated with 1000 mg/kg/day of MPP exhibited moderate tubular basophilia and dilatation, slight tubular casts, minimal tubular necrosis/degeneration, moderate interstitial inflammation of the papilla, minimal to slight mineralisation of the medulla, minimal mineralisation of the cortex and slight transitional cell hyperplasia in the pelvis. These findings were found bilaterally.

Moderate to marked tubular basophilia and dilatation, minimal to slight tubular casts, minimal to slight tubular necrosis/degeneration, slight to marked interstitial inflammation of the papilla, slight to marked mineralisation of the medulla and minimal to slight transitional cell hyperplasia in the pelvis were evident in the kidneys of all females treated with 1000 mg/kg/day of MPP. These findings were evident in both kidneys.

Minimal tubular basophilia/dilatation and minimal tubular casts were present in some males given 150 mg/kg/day of MPP.

Although minimal tubular casts were present in one male and one female control and one male given 15 mg/kg/day of MPP, there was an increased incidence of tubular casts in males and females treated with 1000 mg/kg/day of MPP and an increase in severity in males given 1000 mg/kg/day of MPP.

Minimal focal mineralisation in the medulla was present in one female given 150 mg/kg/day. This was one unilateral focal lesion and in the absence of any other treatment related findings, is not considered to be test article related.


Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

All other histological changes were considered to be unrelated to treatment.


Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related changes at the low dose level (15 mg/kg/day)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The results indicated the kidney as a target organ for toxicity. There were adverse histopathological changes in the kidney in both sexes at 1000 mg/kg/day and males at 150 mg/kg/day. Additionally, at 1000 mg/kg/day there were correlating macroscopic and blood chemistry changes, higher water consumption, and lower bodyweight gain and food consumption values. There were no treatment related changes at the low dose level
(15 mg/kg/day) and this level is considered to be the no-observed-effect-level (NOEL) and by default the no-observed-adverse-effect-level (NOAEL).