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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

Link to relevant study records

Reference

Endpoint:

fertility, other

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other: Non-GLP, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment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

GLP compliance:

no

Limit test:

no

Species:

other: 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 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.

Parental animals: Observations and examinations:

GENERAL CONDITION

HEAMATOLOGICAL 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.

Postmortem examinations (parental animals):

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.

PRELIMINARY STUDY
The female vomited approximately 2.5 hours after dosing 0.5 mg/kg test substance on the fifth day and was slightly ataxic after a dose of 0.6 mg/kg four days later. The male vomited on several occasions; after the twenty-first dose (1.3 mg/kg), the twenty-eight dose (1.5 mg/kg) 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) 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 Croup 4 (1.5 mg/kg) 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 histological abnormalities were observed in the sex organs.

Critical effects observed:

no

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Reproductive effects observed:

no

Conclusions:

No pathological changes were found in the sex organs which could be related to the treatment with the test substance. Under the conditions of this test the NOAEL for fertility is 1.5 mg/kg bw.

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. As no pathological changes were found in the sex organs which could be related to the treatment with the test substance, the NOAEL for fertility is 1.5 mg/kg bw.

Effect on fertility: via oral route

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

In the key study, 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. 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 pathological changes were found in the sex organs which could be related to the treatment with the test substance. Under the conditions of this test the NOAEL for fertility is 1.5 mg/kg bw.

In the supporting study, 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, 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.

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.

Effects on developmental toxicity

Description of key information

Oral: Discriminating dose = 1.25 mg/kg bw, rat, pre-guideline study, Farrell 1970

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Twenty pregnant rats per dose were dosed with 0, 0.25, and 1.25 mg/kg bw test substance on day 6 - 15 of the pregnancy by oral gavage.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: between 100 and 144 g
- Housing: 5 to a cage
- Diet: standard pelleted diet, ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: nonylphenolethyleneoxide 0.1%, sodium salt of sulphated cetyl/oleyl alcohol mixture 0.1%, polyglyceryl ricinoleate 0.1 %, and water to 100 %

Details on exposure:

VEHICLE
The suspensions of 0.1 % (w/v), 0.025 % (w/v), and 0.005% (w/v) were prepared by ball milling the drug into a solution of nonylphenolethyleneoxide 0.1 %, sodium salt of sulphated cetyl/oleyl alcohol mixture 0.1 %, polyglyceryl ricinoleate 0.1 %, and water to 100 % .

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

days 6 - 15 of pregnancy

Frequency of treatment:

daily

Duration of test:

Until one day before parturition or after the weaning period.

Remarks:

Doses / Concentrations:
0, 0.25, and 1.25 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Maternal examinations:

Body weight was monitored

Ovaries and uterine content:

The foetuses removed from the uteri one day before parturition were dissected and closely inspected for soft tissue changes and then submitted for alizarin examination.

Fetal examinations:

Offspring that died before weaning were dissected and then sent for alizarin examination. The young that survived to wean were then killed and examined for soft tissue changes.

Indices:

Stillbirths and resorption rates were observed.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Female rats dosed with 1.25 mg/kg bw test substance failed to gain as much weight as the controls. This was probably due to an anorexic effect.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Dosing the mothers during pregnancy with the test substance had no significant effect on stillbirths or resorption rates. Also litter size and mean weights of the offspring were not statistically different from control values. Alizarin and tissue examinations showed no gross abnormalities due to the test substance dosing and all changes were within the normal limits for this strain of rat. Two foetuses in the control group exhibited mild wrist flexure and one had scoliosis. In the 0.25 mg/kg bw dose group, one offspring had a compressed rib cage. Apart from hydronephrosis no other abnormalities were detected.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of the test no teratogenic effects were observed.

Executive summary:

Twenty pregnant rats per dose were dosed with 0, 0.25, and 1.25 mg/kg test substance on day 6 - 15 of the pregnancy by oral gavage. At 1.25 mg/kg signs of maternal toxicity were observed i.e. lack of appetite and poor maternal weight gain. Under the conditions of the test no teratogenic effects were observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1.25 mg/kg bw/day

Quality of whole database:

non-GLP compliant pre-guideline study, Klimisch 2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Information regarding developmental toxicity is available only for non-vomiting species. It should be noted that these oral dose levels are not likely to be reached in humans due to emesis at ca. 0.03 – 0.11 mg/kg bw.

Twenty pregnant rats per dose were dosed with 0, 0.25, and 1.25 mg/kg bw test substance on day 6 - 15 of the pregnancy by oral gavage to investigate teratogenic potential of the test substance. At 1.25 mg/kg bw signs of maternal toxicity were observed i.e. lack of appetite and poor maternal weight gain. Under the conditions of the test no teratogenic effects were observed in the offspring (Farrell, 1970b).

A similar experiment was performed with rabbits. Twelve pregnant rabbits per group were dosed with 0, 0.25, 0.75, and 1.25 mg/kg bw test substance on day 6 - 18 of the pregnancy by oral gavage. No deformities were observed in the offspring but at high doses the test substance was toxic to the dams resulting in spontaneous abortions, and in one animal six resorptions and no viable foetuses were observed. The two highest doses also caused anorexia (Farrell, 1970b). The observed anorexia is a maternal, adverse effect as a result of the test substance. The spontaneous abortions and resorptions are most likely a secondary effect resulting from the maternal toxicity.

Testing the substance in non-vomiting species will not alter the results as emesis will occur and subsequently, systemic absorption of the test substance will be limited. Therefore further testing with another species is scientifically unjustified. More information on emesis in vomiting species (including humans) is described in chapter 7.2 acute oral toxicity.

Justification for classification or non-classification

Based on the available information regarding reproductive toxicity the test substance is not classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.