Pyrimido[5,4-g]pteridine-2,4,6,8-tetramine, 4-methylbenzenesulfonate, base-hydrolyzed

Endpoint summary

Description of key information

28 d (+14 d recovery), rat, gavage: NOAEL/NOEL >= 1000 mg/kg bw/d (GLP, OECD 407; NOTOX B.V. 2001)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males (mean): 185 - 192 g; females (mean): 154 - 156 g
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany)
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenised to visually acceptable levels.
- DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
- RECOVERY: for 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE PRETREATMENT: The samples (between 501 mg and 558 mg) which were taken from the formulations were weighed accurately into suitable volumetric flasks (25 and 50 ml). The flasks were filled up to the mark with formic acid. In order to dissolve the test substance completely, these solutions were sonicated for 5 minutes. The solutions were further diluted with 50/50 (v/v) Milli-Q water / formic acid to obtain concentrations within the calibration range.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC CONDITIONS: Quantitative analyses were based on the largest peak in the HPLC chromatogram of the test substance. The analytical method used was validated.

Analysis of the accuracy of dose preparations revealed values within the range of 98 % to 103 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

Treatment for 28 days, 7 days per week, two weeks of treatment-free recovery for satellite group.

Frequency of treatment:

Once daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: Mortality/Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 1, 2, 8, 14, 15 and 22

FOOD CONSUMPTION: Yes
- Time schedule: Weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: week 4; Since no treatment-related ophthalmoscopic effects were noted in week 4, no ophthalmoscopic examination was performed in week 6
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- Parameters checked: Erythrocytes count, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets count, Red cell distribution width, Total leucocytes count, Differential leucocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes); Prothrombin time, Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- Parameters checked: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Gamma-glutamyl transferase, Bilirubin (total), Chloride, Cholesterol (total), Creatinine, Glucose, Phosphorus, Protein (total), Protein (albumin, globulin, Albumin Globulin ratio), Triglycerides, Urea, Calcium, Potassium, Sodium

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, of - Adrenal glands, Aorta, Brain (representative regions, including cerebrum, cerebellum and pons), Caecum (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung (infused with formalin), Lymph nodes (mandibular, mesenteric), (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), all gross lesions
- Organ weights: Adrenal glands, Epididymides, Kidneys, Ovaries, Testes, Brain, Heart, Liver, Spleen, Thymus
HISTOPATHOLOGY: Yes, all tissues and organs collected at the scheduled sacrifice from all main group animals of the control and the highest dose group and all gross lesions of all animals (all dose groups).

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. There were no clinical signs of toxicity or behavioural changes during the study period that were considered to be related to treatment.

All group 3 and 4 animals had produced yellow faeces from day 2 of the treatment phase onwards. This finding, which remained present up to day 3 of the recovery period in all high dose animals, was considered to be related to staining properties of the test substance.
Other findings consisted of alopecia and scabs, which are commonly noted in rats of this age and strain, housed and treated under the conditions in this study. These findings were therefore considered of no toxicological significance. Clinical signs were absent among group 2 animals and control females.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals were considered to have been unaffected by treatment with the test substance.

FOOD CONSUMPTION
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings in week 4.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

Individual values comprising the increased mean platelet value of high dose females at the end of treatment were within the range of historical control data. No dose-related response was obtained for the decreased mean corpuscular haemoglobin concentration values of group 2 and 3 animals at the end of treatment, which were also comparable to historical controls. The incidence of alterations of individual white blood cell and neutrophil counts during the in-life phase exceeding the historical control range, was unrelated to the dose. No toxicological relevance was attached to these findings.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with TKP 50052.
The slightly increased bilirubin and chloride values obtained for high dose males at the end of treatment were comparable to controls of similar studies and no microscopic correlates were found. No dose-related response was achieved for statistically significant changes occurring among female groups at the end of treatment. Of these, the increased chloride and decreased urea values were comparable to other studies. Creatinine values of treated males at the end of the treatment phase were slightly low in comparison to similar studies. No explanation could be given for this finding. Changes attaining a level of statistical significance at the end of the recovery phase were not present at the end of treatment and were of a slight nature. Therefore, these alterations were considered to be of no toxicological importance.

ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered to have been unaffected by treatment with the test substance.
Thymus and thymus:body weight ratio showed an apparent increase in high dose females at the end of the recovery phase. Since no such finding was present at the end of treatment, this finding was considered to have occurred by chance and to be without toxicological relevance.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Yellowish discolouration of the cranial lobes of the lung of one high dose male was considered to be related to the presence of macrophages containing yellow-brown pigment. Gray-white discolouration of the cortex of the left kidney of one group 4 female correlated microscopically to a benign nephroblastoma. This lesion was considered to be within the historical background data. Other incidental findings among control or treated animals included yellowish nodules on the epididymides, dark red discolouration of the lungs or mandibular lymph nodes, scab formation on the skin, gray-white foci on the right lateral lobe of the liver, an uterus containing fluid, and dark red foci on the thymus. These findings are occasionally seen among rats used in these types of studies and at the incidence observed they were considered changes of no toxicological significance. Group 2 and 3 males were without macroscopic findings.

HISTOPATHOLOGY
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
Pigmented macrophages (containing brown-yellow pigment) in the lungs of two high dose animals and in the oesophageal wall of one high dose animal were noted. This pigment was considered likely to be the test compound and to be related to the gavage procedure, rather than being a toxicological event. Other microscopic findings were within the range of background pathology encountered in rats of this strain and age. These included minimal focal necrosis in the liver of two high dose group females and an early nephroblastoma in one high dose group female.

OTHER FINDINGS
Functional Observations
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with TKP 50052, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.
Total motor activity of the control males as recorded by both the high and low sensor, and low sensor counts of group 3 and 4 males were slightly low in comparison to historical control data (average high and low sensor activity 2750 and 5965 counts/12 hours respectively (high sensor: n=241; low sensor: n=243)). There were no supportive clinical signs or neurological abnormalities noted. No explanation could be given for these findings, which were considered to be without toxicological implication.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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 subacute oral toxicity of TKP 50052 (purity 94.8 weight-%) in the rat was investigated in a GLP conform study according to OECD guideline 407 with a daily gavage application in the rat for 28 consecutive days, followed by a 14 day recovery period.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; ophthalmoscopy at week 4; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

In none of the treatment groups, treatment related findings were observed.

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) for TKP 50052 of 1000 mg/kg/day was established.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the test item is not considered to be classified for its potential for repeated dose toxitity under Regulation (EC) No. 1272/2008.