2-amino-6-methyl-4-propyl-1,2,4-triazolo[1,5-a]pyrimidin-5(4H)-one

Endpoint summary

Description of key information

Oral: Discriminating dose = 1.5 mg/kg bw/day, male, beagles, pre-guideline study, Farrell 1970

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of each 8 beagles were dosed daily for 3 months with capsules containing 0, 0.15, 0.5, and 1.5 mg/kg bw test substance.

GLP compliance:

no

Limit test:

no

Species:

dog

Strain:

other: beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Thirty-two beagles were randomly divided into groups of four males and four females and maintained throughout the test on the usual stock diet.

Route of administration:

oral: capsule

Vehicle:

other: lactose, maize starch, magnesium stearate

Details on oral exposure:

Tablets test substance were placed in hard gelatin capsules. Tablet composition:
- 0.6 mg test substance, 91 mg lactose, 7.4 mg maize starch, and magnesium stearate 1.0 mg (total tablet weight of 100 mg)
- 5 mg test substance, 180 mg lactose, 13 mg maize starch, and magnesium stearate 2.0 mg (total tablet weight of 200 mg)
- 6 mg test substance, 180 mg lactose, 12 mg maize starch, and magnesium stearate 2.0 mg (total tablet weight of 200 mg)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 0.15, 0.5, and 1.5 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a preliminary test with a male and female beagle dosed daily with increasing doses for the duration of 39 days.
- Post-exposure recovery period: at the end of the three month period one male and one female dosed with 5 mg/kg bw remained undosed for six weeks to assess the reversibility of any lesions.

Observations and examinations performed and frequency:

GENERAL CONDITION

HAEMATOLOGICAL EXAMINATIONS
Blood from each dog was examined before the test began and after one, two and three months of dosing. The examination consisted of haemoglobin estimation, packed cell volume, total white blood cell count, differential white cell count platelets and mean cell haemoglobin.

BONE MARROW FINDINGS
Bone marrow smears from the femoral marrow and samples from the sternal marrow were taken from all dogs at autopsy

URINE ANALYSIS
Urine was collected from each animal before the test began and on days 42 and 91 of treatment.

CLINICAL CHEMISTRY
All dogs were bled for clinical chemical estimations before the test began and on days 40 and 89 of dosing. The investigations made were: blood sugar, serum urea, total protein, electrolytes (Na+, K+, and Ca++), serum alkaline phosphatase activity, serum aspartate amino transferase activity (AST) and serum isocitrate dehydrogenase activity (ICD). In addition to this, the serum enzymes (alkaline phosphatase, AST and ICD were measured in two males and two females of control and top dose group on days 5 , 12 , 16 and 21.

CLINICAL PHARMACOLOGY
Observations were made on arterial blood pressure, heart rate, respiration rate and E.C.G.of all dogs twice before treatment commenced, and after one week and one and three months of dosing.

SERUM LEVEL ASSAYS
Serum concentrations were measured in dogs treated for 41, 55 and 90 days.

Sacrifice and pathology:

AUTOPSIES
An autopsy was performed on every dog. The organs were weighed and samples of tissues put into fixative for histological examination.

HISTOLOGICAL EXAMINATION
The following tissues of each animal were examined: kidneys, adrenals, heart, liver, lung, spleen, pancreas, thymus, lymph nodes (abdominal, thoracic and neck), gonads and accessory sex organs, mammary glands, thyroid, stomach, small intestine, large intestine, bladder, quadriceps muscle, eyes, pituitary, and brain.

Details on results:

PRELIMINARY STUDY
The female vomited approximately 2.5 hours after dosing 0.5 mg/kg bw test substance on the fifth day and was slightly ataxic after a dose of 0.6 mg/kg bw four days later. The male vomited on several occasions; after the twenty-first dose (1.3 mg/kg bw), the twenty-eight dose (1.5 mg/kg bw) and after feeding on the twenty-ninth day. No histological changes were observed in either of the animals.

GENERAL CONDITION
One control female developed eczema on its back and was dosed for five days with 2 mL Streptopen and 1 mL Betsolan. After the fifth week of treatment many of the dogs receiving the highest dose salivated profusely before dosing on several occasions. Also one male in the same group refused to eat on days 9 and 10 of treatment. Vomiting occurred sporadically in six of the animals in the highest dose groups and three in the middle dose group from the ninth day onwards. It usually occurred after dosing but occasionally the dogs vomited before dosing. One female in Group 4 was sick on several occasions and passed blood in the faeces on day 95: she was killed on that day and found to have an ileo-caecal intussusception. Weight gains were similar in the males in all groups, but the females receiving the highest dose lost weight from time to time.

HEAMATOLOGICAL EXAMINATIONS
There were no changes in the haematological values attributable to the administration of the test substance.

BONE MARROW FINDINGS
The female in the highest dose group that was killed and found to have an intussusception showed megaloblatic hyperplasia. This was possibly related to poor intestinal absorption due to the ileo-caecal ulceration.

URINE ANALYSIS
There were no changes attributable to the administration of the test substance.

CLINICAL CHEMISTRY
There were no changes attributable to the administration of the test substance.

CLINICAL PHARMACOLOGY
Respiration rates and E.C.G.s of all animals remained within normal limits. There were no changes attributable to the administration of the test substance.

SERUM LEVEL ASSAYS
The compound is well absorbed after oral administration.

ORGAN WEIGHTS
There were no significant differences between the weights of the various organs in treated and control groups.

HISTOLOGICAL EXAMINATION
There were no changes attributable to the administration of the test substance. At autopsy many of the animals, both treated and control, were observed to have large reddish areas in the lungs. These were patches of pneumonia or nodules of inflammatory cells. Cross sections of nematodes were also present in many sections of the lung. Due to an oversight in animal husbandry the dogs used in this experiment did not have the usual treatment with anthelmintics prior to the start of dosing. All the lesions in the lungs are therefore thought to be attributable to the larval worms. One female receiving the highest dose (1.5 mg/kg bw) had a small cystadenoma in the thyroid, and the female in this group which was killed on day 95 showed ulceration of the colon in the area of intussusception. Intussusception is not uncommon in these dogs. All other changes seen were those normally found in these dogs. At the end of the three month period of dosing one male and one female dog from Group 4 (1.5 mg/kg bw) were left undosed for six weeks to assess the reversibility of any lesions. At the end of the six weeks both dogs were autopsied in a similar manner to the others. No pathological changes attributable to the administration of the test substance were found. Both dogs showed areas of bronchopneumonia and inflammation in the lungs but this was thought to be related to the infestation of the lungs by larval nematodes.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

not specified

Conclusions:

Vomiting occurred in six of the eight animals dosed with 1.5 mg/kg bw test substance and in three of the eight receiving 0.5 mg/kg bw test substance. No other test substance related changes were observed.

Executive summary:

Four groups of each 8 beagles were dosed daily for 3 months with 0, 0.15, 0.5, and 1.5 mg/kg bw test substance via capsules. There were no test substance attributable changes observed with haematological examination, bone marrow examination, urine analysis, clinical chemistry and clinical pharmacology. Vomiting occurred in six of the eight animals dosed with 1.5 mg/kg bw test substance and in three of the eight receiving 0.5 mg/kg bw test substance. No other test substance related changes were observed. Under the conditions of this test the NOAEL is 1.5 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1.5 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

non-GLP compliant pre-guideline study, Klimisch 2

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

Repeated dose

Oral:

The test substance is a known emetic in humans (Bayliss, 1973) therefore tests in vomiting species are most relevant. The study performed in the rat, a non-vomiting species, was therefore considered only as supportive information since the dosed test substance is likely more available for absorption in non-vomiting species compared to vomiting species. The study performed in beagles was assigned as a key study since this is a relevant test species. Although the study is non-GLP compliant and a pre-guideline study, the study is adequate for assessment.

Four groups (n=30 or 20) of rats were dosed daily for 3 months with 0, 0.25, 1.25, and 5 mg/kg bw test substance by oral gavage (Farrell 1970b). Serum level analysis indicated that the substance is well absorbed after oral administration. There were no changes measured attributable to the administration of the test substance with haematological examinations, urine analysis, organ weights and histological examination. No abnormalities related to the administration of the test substance were observed in the levels of aspartate amino transferase, isocitrate dehydrogenase or total protein. Slightly elevated levels of alkaline phosphatase were found in both the male and female treated rats on day 22, but by day 85 these levels had returned to the normal range. Significantly elevated levels of urea were present in the serum of treated female rats on days 36, (p < 0.001) particularly in two females. After 85 days all five females showed elevated levels (p < 0.01); the control females on this day were, however, higher than observed previously in this test. The treated male rats also showed a slight elevation of serum urea levels on day 36 (p < 0.05), but this was not apparent on day 85. The kidneys of the treated rats were normal. Under the conditions of this test the NOAEL is 5 mg/kg bw.

Four groups of each 8 beagles were dosed daily for 3 months with 0, 0.15, 0.5, and 1.5 mg/kg bw test substance via capsules. There were no test substance attributable changes observed with haematological examination, bone marrow examination, urine analysis, clinical chemistry and clinical pharmacology. Vomiting occurred in six of the eight animals dosed with 1.5 mg/kg bw test substance and in three of the eight receiving 0.5 mg/kg bw test substance. No other test substance related changes were observed. Under the conditions of this test the NOAEL is 1.5 mg/kg bw (Farrell, 1970b).

Increasing the dose above 1.5 mg/kg bw will increase the occurrence of emesis and this will limit the systemic absorbance of the test substance. Therefore further testing with a higher dose is scientifically unjustified. More information on emesis in vomiting species (including humans) is described in chapter 7.2 acute oral toxicity.

Inhalation:

No studies are available regarding repeated dose via inhalation. Inhalation followed by absorption of the test substance will result in emesis in vomiting species. Humans are most sensitive to the emetic effects and vomiting occurs at 2 – 8 mg (ca. 0.03 – 0.11 mg/kg bw).The test substance will be more available for absorption via inhalation compared to oral exposure since the dose cannot be reduced by vomiting. However emesis will be induced at similar plasma levels of the test substance independent of the exposure route. Because of great discomfort as a result of emesis it is highly unlikely that exposed subjects will stay in the exposure area, interrupting exposure. Thus in practice the highest systemically available concentration by inhalation exposure that can be reached is similar to that after repeated oral exposure. No substance related adverse effects were observed in vomiting species after repeated oral exposure at non-vomiting concentrations (1.5 mg/kg bw in beagles) and therefore no adverse effects are expected after repeated inhalation exposure below vomiting concentrations.

Justification for classification or non-classification

Based on the observed clinical signs (emesis) up to 1.5 mg/kg bw in the 90 day oral toxicity study in dogs the test substance is not classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.