2-Propen-1-one, 3-(dimethylamino)-1-(3-pyridinyl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 June 2000 - 24 July 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EEC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-770-8

EC Name:

-

Cas Number:

55314-16-4

Molecular formula:

C10 H12 N2 O1

IUPAC Name:

3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one

Details on test material:

- Name of test material (as cited in study report): STI571 Y5A
- Substance type: Brown solid
- Physical state: solid
- Analytical purity: >95%
- Lot/batch No.: 0093800007
- Expiration date of the lot/batch: 08 May 2001 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: In refrigerator in the dark.
- Purity: Not indicated by sponsor; treated as 100% pure
- Stability under storage conditions: Not indicated
- Stability in vehicle: see details on Analytical verification of doses or concentrations.

Test animals

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.
- Fasting period before study: not applicable
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3°C. Deviations from these optimal conditions occurred, but were considered not to have affected study integrity.
- Humidity (%): 30-70%. Deviations from these optimal conditions occurred, but were considered not to have affected study integrity.
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.
Lighting conditions exceeded the protocolled range on some days. Deviation was slight and incidental in nature.

IN-LIFE DATES: From: 26 June 2000 To: 23 July 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(Milli-U)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 200 mg/mL


Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Gavage procedure was conducted using a stainless steel stomach tube.
Formulations were placed on a magnetic stirrer during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 4 formulations were analysed to check stability over 4 hours, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations).

Test substance formulations in Milli-U water were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 96 % to 100 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

At least 28 days.

Frequency of treatment:

Once daily, at approximately the same time each day, 7 days per week. Animals were dosed up to the day prior to necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 294828). See attached file.

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on days 8, 15, 22 and 28, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION: Weekly
- Food consumption for each cage determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m..
- Anaesthetic used for blood collection: Yes (iso-flurane )
- Animals fasted: Yes, overnight
- How many animals: All animals.
- Parameters examined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Platelet count, Red cell distribution width, Total leucocyte count, Differential leucocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes), Prothrombin time, Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m..
- Animals fasted: Yes, overnight
- How many animals: All animals.
- Parameters examined: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Bilirubin, Chloride, Cholesterol, Creatinine, Glucose, Phosphorus, Protein, albumin, Urea, Calcium, Potassium, Sodium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment.
- Dose groups that were examined: all dose groups.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal glands, Aorta, Brain, 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.

Tissues in parentheses in the above list were not examined as there were no signs of toxicity or target organ involvement.

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus.

The following organs were missing: One rectum, Mandibular lymph nodes from one animal, Thyroid/parathyroid from one animal, Sciatic nerve; nerve from head taken instead from 2 animals. Sufficient data remained available for evaluation.

All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY: Yes
Slides of all organs and tissues collected at the scheduled sacrifice from all control, group 3 and 4 animals, as well as from all animals which died spontaneously or were terminated in extremis, and all gross lesions of all animals were examined by a pathologist. All abnormalities were described and included in the report. Tissues in parentheses in the above list under Grss Pathology were not examined as there were no signs of toxicity or target organ involvement.

Slides of all organs and tissues collected at the scheduled sacrifice from all group 3 animals were also examined by a pathologist. Group 2 animals were not examined. Due to high mortality among group 4 animals, it was considered necessary to gain better insight into potential lesions. Group 2 animals were not examined since treatment-related findings in group 3 animals were absent.

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. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY:
Four males and two females of group 4 (1000 mg/kg) died or were sacrificed moribund between treatment days 14 and 26. Before death these animals (except one animal) showed clinical signs indicative of toxicity (see below). Based on macroscopic and microscopic findings these deaths were considered to be related to treatment with the test substance. No unscheduled deaths occurred among group 1, 2 or 3 animals.

CLINICAL SIGNS:
Clinical signs indicative of toxicity were noted among animals dosed at 1000 mg/kg only and included lethargy, muscle twitching, hunched posture, piloerection, emaciated appearance, calm behaviour, and uncoordinated movements among 4/5 males, and hunched and/or cramped posture and emaciated appearance among 3/5 females. These signs were generally noted in week 4 or on the days preceding death only.

Dark yellow discolouration of the urine was observed on the trays beneath the cages containing group 4 animals from day 2 onwards. This was considered to be due to staining properties of the test substance. Post-dosing salivation was noted among all group 4 males and all group 3 and 4 females. This clinical sign is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or irritant or bad taste of the test substance. Alopecia (usually on the back or neck), scabs and brown staining of the fur may be stress-related phenomenons occurring secondary to the treatment procedure. Therefore, these signs were considered not to be a sign of systemic toxicity.
No clinical signs were noted among control animals and animals dosed at 50 mg/kg/day.

FUNCTIONAL OBSERVATIONS:
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with STI571 Y5A, when compared to control animals.
The average total high and low sensor counts of the sole surviving group 4 male were slightly low when compared to control values. Other variations in motor activity did not indicate a relation with treatment.

An increased motor activity (both high and low sensors) was noted in one female (50 mg/kg/day). In the absence of a dose-dependent relationship this was considered to have occurred by chance and to be without any toxicological relevance. For one male (control) high:low ratio scorings per hour were extremely high at several instances. Since the biological relevance of these changes was doubted the high sensor recordings of this animal were excluded from interpretation.

BODY WEIGHTS:
Body weight gain of males dosed at 1000 mg/kg/day was reduced throughout the treatment period, resulting in a reduction of mean body weights of up to 28% when compared to controls.
Among high dose females a reduced body weight gain became apparent in the second half of the treatment period, with mean body weights being reduced with approximately 9%.

Reduced body weights of females dosed at 50 mg/kg in weeks 2 and 4 were without a dose response relationship. Moreover, most individual values remained within the range expected for rats of this age and strain. Therefore, the statistically significant decreased body weights of group 4 females in week 2 was considered not to be an overt sign of toxicity. Body weights and body weight gain of group 3 (150 mg/kg) animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION:
Reduced food consumption and relative food consumption (i.e. after correction for body weight) was noted among males dosed at 1000 mg/kg in weeks 3 and 4 of the treatment period.

Absolute and relative food consumption of females did not show treatment-related alterations.

CLINICAL LABORATORY INVESTIGATIONS:
From group 4 (1000 mg/kg), only one male and three females were available for blood sampling.

HAEMATOLOGY:
The surviving group 4 male showed slightly lower white blood cell and lymphocyte counts, increased neutrophil counts and mean corpuscular haemoglobin concentration values, and a decreased prothrombin time. These values were (slightly) below or exceeded the range of historical data. Mean corpuscular volume and mean corpuscular haemoglobin values were decreased among high dose females (p<0.05). However, all individual values for mean corpuscular haemoglobin remained within the range of historical data.

Other inter-group differences which attained a level of statistical significance were considered not to be related to treatment as they did not form a dose response relationship and all individual values remained within the range of control values found for rats of this age and strain.

CLINICAL BIOCHEMISTRY:
The surviving group 4 male showed higher bilirubin, cholesterol, total protein, sodium and albumin levels, and decreased potassium values, whereas the females of group 4 had higher alanine aminotransferase activities (p<0.01) and bilirubin levels (p<0.05).

The increased inorganic phosphate values as noted among high dose females were considered to be within the range of values that can be expected for rats of this age and strain based on comparable 28-day studies. Other inter-group differences which attained a level of statistical significance did not form a dose response relationship. Therefore, these alterations were considered not to be related to treatment.

MACROSCOPIC EXAMINATION:
Macroscopic findings considered to be related to treatment occurred among animals of group 4 (1000 mg/kg) only and consisted of a reduced size of the prostate, seminal vesicles, spleen and thymus, emaciated occurence (each in 1/5 males) and stomach abnormalities (thickened wall (1/5 males), thickened limiting ridge (2/5 males), irregular surface (1/5 males), crateriform retractions of the glandular mucosa (1/5 males)).

The remainder of the macroscopic findings recorded were considered to be spontaneous in nature and not related to treatment with the test substance.

ORGAN WEIGHTS:
Spleen weight (absolute and relative) of the surviving group 4 male (1000 mg/kg) was reduced.

The increased absolute liver weights and liver:body weight ratios of high dose females might reflect a higher (metabolic) activity in this organ after administration of high doses of a xenobiotic agent and may thus simply represent an adaptive change to treatment, rather than a toxic effect. No relevance was attached to the statistically significantly higher testes weights of group 3 males as the difference lacked a dose response relationship. Similarly, no relevance was attached to the lower absolute brain weight of group 4 females and the slightly reduced weights of the thymus and epididymides of the group 4 male as this was considered to reflect the lower body weight recorded for this group.

MICROSCOPIC EXAMINATION:
A range of microscopic findings were recorded in rats of group 4 (1000 mg/kg) in the following organs*:
- Lungs: alveolar macrophage foci (3/5 ♂)
- Stomach: focal glandular erosion (1/5 ♂), submucosal oedema (1/5 ♂) and squamous hyperplasia (3/5 ♂) of the forestomach [thickened (limiting ridge)]
- Prostate: atrophy (all ♂) [reduced size]
- Seminal vesicles: glandular atrophy (1/5 ♂) [reduced size]
- Thyroid glands: follicular hypertrophy/hyperplasia (1/5 ♂, 2/5 ♀) [reduced size]
- Adrenal glands: diffuse cortical hypertrophy (1/5 ♂)
- Spleen: lymphoid atrophy (4/5 ♂) [reduced size]
- Bone marrow: erythroid atrophy (3/5 ♂, 1/5 ♀), myeloid hyperplasia (3/5 ♂, 2/5 ♀)
- Thymus: increased lymphocytolysis (2/5 ♂), lymphoid atrophy (all ♂, 1/5 ♀) [reduced size]
* Amongst these findings were histologic correlates to the macroscopic observations, mentioned between [straight brackets].

The remainder of the microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and group 3 (150 mg/kg/day) rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 150 - < 1 000 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Toxic effects were only noted at 1000 mg/kg. Rats treated at 50 or 150 mg/kg/day were free of treatment-related findings.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with STI571 Y5A by daily oral gavage resulted in the death of four males and two females at 1000 mg/kg. Various clinical signs, macroscopic and microscopic findings were noted among these animals, which indicated that the cause of death or moribundity in these rats may be due to poor overall condition and stress as a result of treatment with the test substance.
 
Toxic effects were only noted at 1000 mg/kg, and were most overtly present among males. These effects consisted of reduced body weights, associated with a reduced food intake. Reduced terminal body weights may reflect the decreased weights of several organs, such as the decreased splenic weights of females. In addition, laboratory investigations revealed altered values such as a decreased mean corpuscular volume and increased alanine aminoacyl transferase activities among females. The sole surviving male showed several altered blood parameters. Microscopic examination showed adverse morphologic alterations in several organs at 1000 mg/kg which generally confirmed the macroscopic abnormalities. The microscopic findings were considered indicative of general systemic toxicity and stress effects rather than specific organ toxicity and, again, were mainly noted among males.
 
Rats treated at 50 or 150 mg/kg/day were free of treatment-related findings.
 
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for STI571 Y5A of 150 mg/kg/day was established.