Phosphorous ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-04 to 2009-03-04

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

esterification products of castor oil and polyphosphoric acids

EC Number:

614-406-6

Cas Number:

68308-61-2

Molecular formula:

C57H107P3O18

IUPAC Name:

esterification products of castor oil and polyphosphoric acids

Constituent 2

Reference substance name:

Polyphosphoric acids, esters with caster oil

IUPAC Name:

Polyphosphoric acids, esters with caster oil

Details on test material:

- Name of test material (as cited in study report): GARDO TP10451
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 2939R
- Expiration date of the lot/batch: 2010-02-18

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
Species and strain: CRL:(WI) BR Wistar rats
Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of strain: Wistar rat is one of the standard species used in rodent toxicity studies
Number of animals: 20 males, 20 females (5 animals/sex/group, 4 groups) and 3 spare rats/sex; at the completion of the study, the spare animals were released to the spare colony, as their use was not required.
Sex: Male and nulliparous, non pregnant female
Age of animals: Young adult rats, less than 9 weeks old
Body weight range at starting: The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight for
each sex (males 240-268 g, females 180-196 g)
Acclimatisation time: 7 days
Animal Husbandry
Animal health: Only animals in acceptable health condition certified by the veterinarian were used for the test.
Cage type: Type III. Polypropylene/polycarbonate
Bedding: Laboratory bedding
Housing/Enrichment: Group caging (5 animals/cage); the animals were housed with more than one animal per cage to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.1 -24.2 C
Relative humidity: 30 – 69 %
Ventilation: 15-20 air exchanges/hour by central air-condition system
The temperature and relative humidity were recorded and checked twice daily during the study. No deviations from the intended ranges were noted
during the study.
Food and Water Supply
Animals received ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice – breeding and maintenance” produced by ssniff Spezialdiäten
GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum.
The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose of integrity of the study. Copies of the relevant Certificates of Analysis are available in the archive at LAB Research Ltd.
Preparation of the Animals
Animals were individually identified using tattoo numbers on the tail, which were cross referenced with LAB Research Ltd’s master file number for each animal allocated to the study, as indicated in the Experimental Design section. The cages were marked by identity cards, with information about
study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

Vehicle/Control Item
Name: Polyethylene glycol 400 (PEG400), Ph. Eur
Batch No.: 1402668
Expiry date: 31 January 2010
Manufacturer: Fluka / Sigma-Aldrich
Storage: At room temperature
Preparation of the Test Item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in vehicle at concentrations of 12.5, 62.5, and 250 mg/mL in the Central Dispensary of LAB Research Ltd. Formulations were prepared at the appropriate frequency to allow their use within 96 hr pending refrigerated storage, according to stability assessment results.
Analytical control (concentration, homogeneity) of formulations was performed in the Analytical Laboratory of LAB Research Ltd. During the first and last weeks of treatment. The dose formulations were homogenous. The measured concentrations ranged from 97 to 99% (first week of the treatment), and from 94 to 108 % (last week of the treatment) of the nominal concentrations (12.5, 62.5, and 250 mg/mL). No test item was detected in the control samples. Assessment of test item stability in this vehicle, under the conditions employed on the study (LAB study code 08/625-316AN) indicated up to 6-hour stability at room temperature; in addition, Gardo TP10451 in PEG400 formulations at concentrations from approximately 1 to 250 mg/mL, stored refrigerated (5±3°C), proved to be stable for up to 96 hours, with a recovery within the acceptable range of 100 ± 10% (104-94 %).

Duration of treatment / exposure:

Duration of treatment: 28 days.

Frequency of treatment:

The test item was administered by oral gavage on a 7 days/week basis.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
12.5
Basis:
other: administed by gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
62.5
Basis:
other: administed by gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250
Basis:
other: administed by gavage

No. of animals per sex per dose:

20 males and 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

Justification of the dose selection:
Dose levels were selected based on available information, as the acute oral toxicity study (acute toxic class method, LAB Study Code: 08/625-001P), and 14-day oral dose range finding toxicity study in CRLWI)BR rats (LAB study no.: 08/625-100PE). In the acute study, Gardo TP10451 caused no mortality at 300 or 2000 mg/kg bw dose level. Although sunflower oil was used as the vehicle in the acute oral toxicity study, PEG 400 has been selected as a suitable vehicle for the 14-day preliminary study, based on the analytical method used. In the 14-day preliminary study conducted at dose levels up to and including 1000 mg/kg bw/day, no mortality or adverse effects were observed clinically, and no test item related changes occurred in animal body weight, food consumption, or clinical pathology parameters. No macroscopic findings were noted at the completion of the treatment.
The oral route was selected as a probable route of human exposure.

Positive control:

NA

Examinations

Observations and examinations performed and frequency:

Clinical Examination
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, at approximately the same time in the mid morning hours. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, unusual respiratory pattern), occurrence of secretions and excretions, circulatory and central nervous system, somatomotor activity and behaviour pattern (clonic or tonic movements, stereotypies, bizarre behaviour), changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma; no such clinical signs were noted during the study. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were assessed in all animals in the fourth exposure week (Day 26). General physical condition
and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968). Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Weight Assessment
For each animal body weight was recorded on Day 0 (beginning of the experiment), then weekly during treatment period with precision of 1 g. On the day of gross pathology, fasted body weights were determined.
Food Consumption
The food consumption was determined weekly by re-weighing the non consumed diet during the treatment period with a precision of 1 g.
Clinical Pathology
Laboratory examinations (haematology and clinical chemistry) were conducted at the end of the treatment period (Day 28). After an overnight food deprivation (approximately 16 hours), animals were anaesthetised with Euthasol 40% and blood samples were collected by cardiac puncture. Three samples were taken from each animal: one for haematology (tubes contained K-EDTA 1.6 mg/mL blood as anticoagulant), one for determination of blood clotting times (APTT and PT; tubes contained sodium citrate as anticoagulant) and the third one to obtain serum samples (tubes did not contain any anticoagulant) for clinical chemistry determinations.
Haematology
The following haematology parameters were assessed in all animals:
-RBC Red Blood Cell (erythrocyte) count, (1012/L) M/L
-Hgb Haemoglobin concentration, (g/dL)
-Hct Haematocrit (relative volume of erythrocytes) (%)
-MCV Mean Corpuscular (erythrocyte) Volume (fL)
-MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
-MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
-RDW Red Cell (erythrocyte) volume (%)
-Plt Platelet (thrombocyte) count, (109/L) K/L
-MPV Mean Platelet Thrombocyte volume (fl)
-RETIC % Reticulocyte count (%)
-WBC White Blood Cell (leukocyte) count, (109/L) K/µL
-NE % Neutrophil (%)
-LY % Lymphocyte (%)
-MO % Monocyte (%)
-EO % Eosinophil (%)
-BA % Basophil (%)
-LUC % Large Unstained Cells (%)
-APTT Partial Thromboplastin Time (sec)
-PT Prothrombin Time (sec)
Clinical Chemistry
The following clinical chemistry parameters were assessed in all animals:
-Glucose Blood sugar conc. (mmol/L)
-T-BIL Total Bilirubin conc. (μmol/L)
-Urea Urea conc. (mmol/L)
-Chol. Cholesterol conc. (mmol/L)
-Creat. Creatinine conc. (μmol/L)
-Phos. Phosphorus conc. (mmol/L)
-Na+ Sodium conc. (mmol/L)
-K+ Potassium conc. (mmol/L)
-Ca++ Calcium conc. (mmol/L)
-Cl Chloride conc. (mmol/L)
-Tot. prot. Total Protein conc. (g/L)
-Alb. Albumin conc. (g/L)
-A/G Alb/glob ration
-AST Aspartate Aminotransferase activity (U/L)
-ALT Alanine Aminotransferase activity (U/L)
-ALKP Alkaline. Phosphatase –activity (U/L)
-GGT Gamma Glutamyltransferase activity (U/L)
-Bile acids (µmol/L)
Organ Weight
The following organs were weighed and recorded. Paired organs were weighed together.
With precision of 0.01g: Liver, kidneys, testes, epididymides, prostate + seminal vesicle with coagulating glands as a whole, uterus, including cervix, thymus, spleen, brain and heart.
With precision of 0.001g: Adrenals, ovaries

Sacrifice and pathology:

Gross pathology was performed on all experimental animals, euthanized at termination under pentobarbital anaesthesia, followed by exsanguination.
After exsanguination the external appearance was examined, cranium,thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size. The following organs/tissues were removed and preserved in 10% buffered formalin solution for histological processing: Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum and jejunum with Peyer’s patches, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, ovaries with oviduct, uterus with cervix, vagina, epididymides, brain (including cerebrum, cerebellum, pons and medulla oblongata), lachrymal gland with Harderian glands, seminal vesicle with coagulating glands, muscle (quadriceps), sciatic nerve and aorta. The eyes with the optic nerves and the testes were preserved in modified Davidson fixative for histological processing.
Histopathology
Full histopathology was performed on the preserved organs or tissues of the animals of the control group and Group 4. In Groups 2 and 3, the tissues with gross lesions were also processed histologically. As no toxicologically significant microscopic findings were noted in the examined organs and tissues in Group 4 (High dose), no additional histopathological evaluation is considered required; however, the need to further microscopic examination on organs in the mid and low dose group will be decided after consulting the Sponsor; future results, if any, will be reported.

Other examinations:

NA

Statistics:

Statistical analysis was performed on the following data sets using SPSS PC+ software:
- body weight
- haematology
- clinical chemistry
- organ weight
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.Where significant heterogeneity was found, the normal distribution of data was assessed by a Kolmogorov-Smirnov test. In case of a not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the intergroup comparisons were performed using a Mann-Whitney U-test.
Frequency of clinical observations, mean daily food consumption by groups and sex, frequency of pathological and histopathological findings by sex and dose were calculated.

Results and discussion

Results of examinations

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:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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:

no effects observed

Details on results:

Mortality
No mortality occurred during the 28-day treatment period.
Clinical Observations
Daily and Detailed Weekly Observations
There were no clinical signs observed following administration of Gardo TP10451 by oral gavage, daily for 28 days, at dose levels up to and including 1000 mg/kg bw/day.
Functional Observation Battery
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment period.
Minor changes were observed in the animals throughout all the dose groups,including controls when subjected to the modified Irwin test (functional
observation battery). However, the variations noted demonstrated no treatment-related differences to the control, and were considered within the
normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Incidences and extent of reactions observed in the treated animals were comparable to those observed in control animals and are without any toxicological significance.
Oestrus Cycle Evaluation
Evaluation of the vaginal smears on Day 28 prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats.
Body Weight
No adverse effects were noted on the mean body weight and body weight gain values in the treated groups compared to control animals following daily administration of Gardo TP10451 at dose levels of up to and including 1000 mg/kg bw/day.
Slightly higher mean body weight gain values than control were noted, with statistical significance in the male animals at all the dose levels tested (Day 21-27). In the absence of a consistent dose or gender-related response, these changes were considered neither toxicologically significant, nor related to Gardo TP10451 administration, but expected variations in the population of Wistar rats.
Food Consumption
There were no test item related differences in the mean daily food intake in any test item treated groups (50, 250, or 1000 mg/kg bw/day, male or female) when compared to the control.
Haematology
There were no toxicologically relevant differences between the control and test item-treated groups, or any adverse effects of Gardo TP10451 on
haematology parameters in the male and female animals.Statistically significant variations were noted on occasion in the values of a few haematology parameters. In the male animals, lower mean large unstained cells (LUC) values were observed at 50 mg/kg bw/day. In the female animals, the mean values of haemoglobin (Hgb, at 250 and 1000 mg/kg bw/day), haematocrit (Hct, at 1000 mg/kg bw/day), platelet thrombocyte volume (MPV, at 50, 250, and 1000 mg/kg bw/day) and neutrophil (NE%, at 50 mg/kg bw/day) showed variations compared to control animals.Evaluation of the mean and individual results in correlation with the control and historical haematology data did not reveal any test-item related cause of these variations. No consistent response was observed in correlation with the dose level, or between male and female animals. The differences observed between the control and treated groups were considered to be incidental or individual findings, which were not related to treatment and generally emained within the historical control ranges, or were with no toxicological significance.
Clinical Chemistry
There were no relevant changes in the examined clinical chemistry parameters that could be attributed to Gardo TP10451 administration.
Statistically significant changes of glucose, concentrations of sodium (Na+), chloride (Cl-), calcium (Ca++), and/or phosphorus (Phos.), aspartate
aminotransferase activity (AST) and alanine aminotransferase activity (ALT), and total bilirubin (T-BIL) were noted in the treated animals compared to
control. In the males, these changes consisted of slightly higher Na+ and Cl- mean values at 1000 mg/kg bw/day and slightly higher Ca++ values than control at all the dose levels tested, with no dose response. In the females, the glucose values were slightly higher than control, statistically significant at 50 and 250 mg/kg bw/day, but well within the historical control range of this laboratory. Minor other variations were noted at 50, 250 and/or 1000 mg/kg bw/day dose levels. These included, but were not limited to, slightly higher Na+, Cl- and/or Phos. mean values, lower AST, ALT and/or T-BIL mean values, compared to control values. The variations were generally within the clinical chemistry historical control range for the population of Wistar rats at this laboratory. Although occasionally these changes were statistically significant, no dose related response was observed, and/or there was no consistent reaction in the male and female groups. These changes were neither considered toxicologically significant, nor indicated a test item related etiology.
Necropsy
There was no evidence of Gardo TP 10451-related macroscopic findings at a dose level up to 1000 mg/kg/bw/day.
Dark discoloration/red of the lungs observed across all groups was regarded as related to terminal procedure. Dark discoloration/red and/or small seminal vesicle, pelvic dilatation and/or mottled kidneys, pale focus/mottled and/or small liver, small testes and spleen, thymic red discoloration/, cyst of the ovary and/or uterus, additionally uterus in estrous cycle were considered to be incidental due to the low incidence and severity.
Organ Weight
There were no statistically, or toxicologically significant changes in the examined organ weight values (absolute and relative to the body and brain weights) noted after Gardo TP10451 administration daily for 28 days, at up to and including 1000 mg/kg bw/day.
Histopathology
There was no evidence of Gardo TP 10451-related microscopic findings at a dose level of 1000 mg/kg/bw/day. Minimal congestion and/or hemorrhage of the lungs observed in all groups were regarded as procedural and correlated with macroscopic observations. Extramedullary hematopoiesis of the spleen and/or adrenal gland, dilatation of the renal pelvis, mononuclear infiltrate in the prostate, congestion and/or hemorrhage in the thymus were regarded as incidental. In addition, deposit of brown cytoplasmic/intranuclear pigment noted in the liver, adrenal gland, sciatic nerve, stomach and cecum in control and/or dosed animals were likely related to histopathological procedure. In summary, Gardo TP10451 administered by oral gavage daily for 28 days to male and female rats, at dose levels up to and including 1000 mg/kg bw/day, did not cause any toxic or other test item related lesions detectable by histological examination of investigated organs in the experimental animals.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Gardo TP10451 administered daily by oral gavage for 28 days in Wistar rats did not lead to any toxicologically adverse effects at dose levels of 50, 250 or 1000 mg/kg bw/day. There were no changes in the animal clinical condition, body weight or food consumption. No Gardo TP10451-related effects were noted at clinical pathology evaluation (haematology, coagulation and clinical chemistry). There were no macroscopic or microscopic findings, or changes in the organ weights that could be ascribed to test item administration.In conclusion, under the conditions of this study, the no observed effect level (NOEL) for Gardo TP10451 is considered 1000 mg/kg bw/day.

Executive summary:

A 28-day oral toxicity study was performed with Gardo TP10451 in male and female CRL:(WI) BR Wistar rats. Dose levels administered in this study were selected from a preceding 14-day dose-range-finding toxicity study performed in the same strain of rats (LAB study code: 08/625 -100PE). A control and three dose groups (n= 5 animals per group and sex) were involved in the study. The test item was administered in concentrations of 0, 12.5, 62.5, and 250 mg/mL prepared in PEG 400 corresponding to 0, 50, 250 and 1000 mg/kg bw/day doses levels, at a 4 mL/kg bw treatment volume. Stability and homogeneity of test item in this vehicle was analytically proven. Assessment of test item stability in this vehicle, in the conditions employed on the study (LAB study code 08/625-316AN) indicated up to 6 hr stability at room temperature, and 96 hr stability refrigerated (5±3°C), at concentrations from approximately 1 to 250 mg/mL in PEG400 formulations, with a recovery within the acceptable range of 100 ± 10% (actual range 104-94% of nominal). Analyses of dose formulations (concentration and homogeneity) were conducted during the first and last week of treatment. The measured concentrations ranged from 94 to 108% of nominal concentrations (12.5, 62.5, and 250 mg/mL). These results were considered suitable for the study purposes. Clinical observations for signs of ill health or reaction to treatment were made once daily. A detailed clinical examination was made before the first treatment, then once weekly during the study with the examinations performed with animals removed from the cage. A functional observation battery was conducted during the last week of the treatment period (Day 26). Body weight and food consumption were measured weekly. Clinical pathology examinations and gross necropsy were conducted at the end of the treatment period (Day 28). The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, and thymus were determined. A full histopathological examination was performed on the selected list of preserved organs and tissues of the animals of Groups 1 (Control) and 4 (High dose), and on abnormal tissues from Low and Mid dose groups.

Results

Gardo TP10451 caused no mortality at up to and including 1000 mg/kg bw/day in CRL:(WI)BR rats. There were no clinical signs following administration of Gardo TP10451 by oral gavage, daily for 28 days. The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative for neurotoxicity were observed. There were no toxicologically significant changes in body weight, body weight gain or animal feed intake between the control and test item treated groups. Minor variations were noted in the clinical pathology parameters (haematology, coagulation and clinical chemistry). Although occasionally these changes were statistically significant, no dose related response was observed, the results were within the historical range, and/or there was no consistent reaction in the male and female groups. These changes were neither considered toxicologically significant, nor indicated a test item related etiology. There were no macroscopic, or microscopic adverse findings, and no statistically, or toxicologically significant changes in organ weight values, or any changes that could be ascribed to test item administration.