1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Marshall BioResources, North Rose, New York
- Age at study initiation: 6-7 months
- Weight at study initiation: males: 6.6 - 8.6 kg, females: 5.4 - 7.2 kg
- Fasting period before study: no
- Housing: Individually housed in stainless steel runs with a flat-slatted fiberglass floor providing a 12.0 square foot area.
- Diet (e.g. ad libitum): Harlan Teklad Global 25% Protein Certified Dog Diet #2025C, ad libitum, except when animals were fasted prior to bleeding.
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-29
- Humidity (%): 30-70
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated once a week in 0.5% aqueous methyl cellulose and stored in a refrigerator. The volume of the dosing mixture each dog received was based on the body weight for each animal at the beginning of each study week, so the dose volume was 1 or 5 mL/kg. The dosing solutions were prepared without correcting for the AI in the test material. The formulated test material was constantly stirred prior to drawing the dose into the dosing syringe. Following administration of the control or test substance to each animal, the gavage tube was flushed with 3 mL of 0.5% aqueous methylcellulose.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 1, 3, and 40 mg/mL (0.95, 3.16, and 38.5 mg/mL analytical)
- Amount of vehicle (if gavage): 1 (on Study Days 0-22) or 5 mL/kg bw (beginning on Study Day 23)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material, mixed in 0.5% aqueous methyl cellulose at 1, 3, and 40 mg/mL, was analyzed for homogeneity. Five samples of each dose suspension were taken and analyzed. The mean concentrations were 0.95 mg/ml (95%, SD=0.01%) for the 1 mg/mL dose suspension, 3.16 mg/mL (105%, SD=0.05%) for the 3 mg/mL dose suspension, and 38.5 mg/mL (96%, SD=0.73%) for the 40 mg/mL dose suspension. Based on the %RSD values of 1.0, 1.7, and 1.9 for the 1, 3, and 40 mg/mL dosing suspensions, the dose suspensions of the test material used in this study were considered homogeneously distributed.
For room temperature stability, samples were analyzed on day 0, 1, and 7. After 7 days, there was no decline in concentration for the 1, 3, or 40 mg/mL dose suspensions. The test material, mixed in 0.5% aqueous methyl cellulose at 1, 3, and 40 mg/mL, was stable at room temperature for a minimum of 7 days.
The concentration of the active ingredient in the dosing solutions was verified for weeks 1, 2, 3, for the 7.5, 15, and 30 mg/mL dose suspensions and for weeks 7, 12, and 13 for the 1.5 and 3.0 mg/ml dose suspensions. The mean concentrations for the study were 89 to 103% of the nominal levels.

Duration of treatment / exposure:

91-92 days except for high-dose group dogs; high-dose group dogs were dosed for a maximum of 36 days

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for this study were based on a 90-day dose-range-finding study with the test material. In that study, compound-induced seizures occurred in both sexes at a dose of 50 mg/kg bw/day.
- Rationale for animal assignment (if not random): The dogs were randomly assigned to dose groups, based on weight, using INSTEM DATATOX. Weight variation of animals used were targeted not to exceed ±20% of the mean weight for each sex. All dogs arrived at the test facility with a supplier's identification number tattooed on the inner part of the ear. This unique identifier was cross-referenced with the unique identification number assigned to each animal.
- Rationale for selecting satellite groups: No satellite groups were included.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily except once daily on weekends and holidays
- Cage side observations included: clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at study initiation and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured weekly throughout the study. Body weights were also taken immediately prior to necropsy to allow for calculation of organ-to-body weight ratios.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes, individual food consumption measured daily throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following the acclimation period and prior to initiation of dosing.
- Dose groups that were examined: Ophthalmic examinations were conducted on all animals, and also on all animals sacrificed just prior to termination of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-exposure and during study weeks 5, 9, and 13.
- Anaesthetic used for blood collection: No, via jugular venipuncture
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Hematocrit (HCT), Hemoglobin (HGB), Leukocyte count (WBC), Erythrocyte count (RBC), Platelet count, Blood clotting measurements (Thromboplastin time, Prothrombin time), Leukocyte differential count, Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin conc. (MCHC), Mean corpuscular volume (MCV), Reticulocyte count, Blood cell morphology, Red Blood Cell Distribution (RDW), Hemoglobin Distribution Width (HDW)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-exposure and during study weeks 5, 9, and 13.
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: All animals.
- Parameters examined: Electrolytes: Calcium (calc), Chloride (Cl), Phosphorus (Phos), Potassium (K), Sodium (Na); Enzymes: Alkaline Phosphatase (ALK), Creatine phosphokinase (CK), Lactic acid dehydrogenase (LD), Alanine aminotransferase (AST/SGOT), Gamma Glutamyl transferase (GGT); Other: Albumin (ALB), Creatinine (Creat), Urea nitrogen (Urea-N), Total Cholesterol (Chol), Globulins (Glob), Glucose (gluc), Total bilirubin (T-Bili), Total protein (TP), Triglycerides (Trig), Uric Acid (Uric-A), A/G ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-exposure and during study weeks 5, 9, and 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Appearance, Volume (UVol), Specific gravity / osmolality (Sp.Gr.), pH, Sediment (microscopic), Protein (Pro), Glucose (Glu), Ketones (Ket), Bilirubin (Bil), Blood (Bld), Nitrite (Nit), Urobilinogen (Uro), Leukocytes (U-Leu)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, a complete gross examination was performed on all animals. The necropsy consisted of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Organs/tissues examined: Cecum, Colon, Duodenum, Esophagus, Gall bladder, Ileum, Jejunum, Liver, Pancreas, Rectum, Salivary glands, Stomach, Larynx, Lung, Nasopharynx, Trachea, Aorta, Bone marrow, Heart, Lymph node (mesenteric, retropharyngeal), Spleen, Thymus, Cervix, Epididymides, Fallopian tube (oviduct), Kidneys, Mammary gland, Ovary, Prostate, Testicle, Ureter, Urinary bladder, Uterus, Vagina, Adrenal gland, Thyroid (with parathyroid), Brain Cerebellum, Cerebellum-Midbrain, Medulla/Pons), Eyes, Nerve (optic, sciatic), Pituitary, Spinal cord (cervical, thoracic, lumbar), Bone (rib/cc jct, sternum), Gross lesions, Muscle, Physical Identifier, Skin
HISTOPATHOLOGY: Yes, all gross lesions, prostate gland and liver from all males; brain, spinal cord, and sciatic nerve from both sexes at all dose levels, and all tissues collected during necropsy in both sexes from the control, 15, and 30 mg/kg bw/day dose levels were examined micropathologically by a veterinary pathologist with the exception of the physical identifier and vagina. Tissues were processed routinely and stained with hematoxylin and eosin (H & E). In this study, selected sections of brain, spinal cord, and sciatic nerve were stained with Luxol Fast Blue (LFB)/Cresyl Violet (CV) and Sevier Munger silver stains. Recuts were requested as deemed necessary. Where appropriate, all findings were assigned a severity score where N = tissues within normal histological limits, 1 = normal, 2 = minimal, 3 = mild or slight, 4 = moderate, 5 = marked, 6 = severe, and 7 = present.The mean severity was determined by dividing the sum of the individual animal severity scores by the number of tissues examined in the group.

Other examinations:

Organ weight: Liver, Lung, Heart, Spleen, Thymus, Epididymides, Kidneys, Ovary, Prostate, Testicle, Uterus, Adrenal gland, Thyroid (with parathyroid), Brain, Pituitary

Statistics:

Statistical significance was determined at p≤0.05 for all tests with the exception of Bartlett's test, in which a probability value of p≤0.001 was used. All tests were two-tailed, except for gross and histopathological lesion evaluations which were one-tailed. Continuous data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Student's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups. Frequency data, that were examined statistically, were initially analyzed by a Chi-Square procedure. If there was statistical significance using the Chi-square test, each treatment group was compared to the control group using a Fisher's Exact test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

mortality observed, treatment-related

Description (incidence):

adverse

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

not compound-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adverse

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
30 mg/kg: seizures in 1/4 males and 2/4 females. These females further showed agressive behaviour, tremors, ataxia, sluggish pupils. One of these females additionally demonstrated laboured breathing, the left pupil did not react to light and the right one was sluggish; the other one showed decreased activity, circling, and both pupils were sluggish. Seizures occurred on study days 15, 22, and 35, respectively. All three dogs were euthanised in-extremis upon observing the seizures.
Due to the seizures, dosing of the 30 mg/kg dose group was stopped on study day 35 and all 30 mg/kg dose animals were euthanised on study day 36.

No animals were found dead.

BODY WEIGHT AND WEIGHT GAIN
There was no compound-related effect on body weight.

FOOD CONSUMPTION
There was no compound-related effect on food consumption. Sporadic statistically significant differences in food consumption for treated groups compared to the control groups were considered incidental.

OPHTHALMOSCOPIC EXAMINATION
No compound-related abnormalities were observed.

HAEMATOLOGY
There were no compound-related hematological findings.

There were few statistical or non-statistical trends in the hematological parameters evaluated in males and females throughout the study, but none were attributed to test-article administration due to a lack of a dose response or consistency of change over the study duration. Examples of these changes comprised:
Males: Statistical increase in prothrombin time of the 15 mg/kg dose group males on day 28.
Females: Statistically increased hematocrit in all dosed females on day 28, accompanied by non-statistical increase in hemoglobin; statistical increase in hemoglobin in the 7.5 and 15 mg/kg dose group, associated with a non-statistical increase in the hematocrit on day 56; activated prothrombin time was statistically increased in the 7.5 mg/kg dose group on days 28 and 56, but similar to pre-treatment values. There were no statistical changes in erythrocyte indices and no biologically important changes in the leukogram (only a few random lymphocyte or monocyte statistical alterations unrelated to dose and similar to pre-treatment values).

CLINICAL CHEMISTRY
There were no compound-related clinical chemistry findings.

There were numerous statistical or non-statistical notations, most occurring in females from the pre-treatment and 28-day evaluation intervals. For example, statistically increased albumin values in 7.5 and 15 mg/kg dose group females at 28 days were similar to the pre-treatment values. Cholesterol was statistically increased in 7.5 mg/kg dose group females at 28 days, non-statistically increased at 56 days, but a similar change was not seen in the 15 mg/kg dose group.

URINALYSIS
There were no compound-related urinalysis findings.

In males, there was a statistical increase in urine volume in both treatment groups (7.5 and 15 mg/kg dose groups) at day 55, but no change at days 28 or 85, and values were similar to the pre-treatment urine volume variability.

In females, there was a statistical increase (but not a biological increase) in protein at all doses in a dose-related manner (values were 15-25 mg/100 mL). However, these changes did not persist throughout the study, were within the individual animal variability, were within mean historical ranges, and the absolute values were in the ranges of most concurrent controls in this study.

ORGAN WEIGHTS
There were no compound-related effects on organ weights.

In females, there were no statistical alterations in either the absolute or relative organ weights.

In males, there was a statistical increase in the 7.5 mg/kg dose group prostate gland absolute weights. This was interpreted to be associated with the variability of sexual maturation at this age and there were no test-compound-related histo-morphologic changes. There was a statistical increase in the absolute liver weights of 7.5 and 15 mg/kg dose group males which was associated with a non-statistical increase in the terminal body weights, a non-statistical and non-dose-related relative liver weight increase, and no definitive histo-morphologic changes in the liver sections evaluated. Therefore, these liver weight changes were not adverse and were interpreted as being incidental and unrelated to test-substance administration.

GROSS PATHOLOGY
There were no compound-related gross pathology findings.

Incidental gross observations included but were not limited to pituitary cyst, spleen zone, reduced-size thymus, thyroid, or prostate, or increased uterus size. The observation of reduced prostate in the 30 mg/kg dose group was associated with the variability of sexual maturation and the early termination of the 30 mg/kg dose group due to seizures in some animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Compound-related morphologic changes:
Observed in the nervous system, characterized as an axonal degeneration particularly within fibers of the dorsal sensory tracts (fasciculus cuneatus) which is the lateral portion of the dorsal spinal cord. There was an associated degeneration and dissolution of myelin in myelinated nerve fibers with phagocytosis of fragmented axonal and myelin debris by macrophages (gitterzellen) producing so-called “digestion chambers”. Hypertrophic reactive astrocytes were noted in some affected regions. Special stains (Sevier-Munger; LFB/CV) were utilized to characterize the lesion, the distribution of which was not typical for known distal axonal lesions. Within the sciatic nerve there was a dose-related increased incidence of generally multifocal axonal degeneration with limited “digestion chamber” formation. This morphologic change was noted in 15 and 30 mg/kg dose group males and females, but the extent of the lesion was subtle in comparison to those in the spinal cord. These test-substance-associated axonal changes were also most subtle in the brain stem affecting morphologic changes in selected animals.

Non-Test-Substance-Related (Background) Lesions:
Within the nervous system there were occasionally observed background changes, generally involving a single individual nerve fibre, with a focal minimal to mild macrophage response (coded in the study as degeneration, nerve fibre), random swollen eosinophilic axon, focal gliosis, or focal lymphocytic or chronic inflammation.

There were other incidental lesions that included renal cysts or inflammation, reduced size or immaturity of the prostate in 30 mg/kg dose group males which were terminated early in the study, and perivascular infiltrate in the liver or lung. The incidence was increased in both sexes of the 30 mg/kg dose group compared to the control group (Males: control 2, dose group 4; Females: control 3, dose group 4; intermediate doses were not evaluated due to the background nature of the change). The change was morphologically incidental, non-statistical, non-clinical relevant, and the severity grade was only slightly increased in 30 mg/kg dose group females. Perivascular infiltrate in the lung was generally minimal to mild, characterized by a generally mononuclear cell presence with occasional macrophages. There were several missing gallbladder sections in this study but due to the absence of lesions in those present, this tissue collection oversight did not affect study interpretation.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

central nervous system

Organ:

spinal cord

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Histopathological test substance-related changes:

	Males				Females
Dose level (mg/kg bw/d)	control	7.5	15	30	control	7.5	15	30
Brain
Axonal swelling	0	1 (1.0)	0	1 (1.0)	0	1 (1.0)	3 (1.0)	1 (1.0)
Axonal degeneration	0	0	0	0	0	0	1 (1.0)	2 (1.5)
Nerve fibre degeneration	0	0	0	0	0	1 (1.0)	0	0
Tissues examined	4	4	4	4	4	4	4	4
Sciatic nerve
Axonal degeneration	0	0	1 (2.0)	1 (2.0)	0	0	2 (1.0)	2 (1.5)
Tissues examined	4	4	4	4	4	4	4	4
Spinal cord
Axonal swelling	0	1 (1.0)	2 (1.0)	0	0	0	0	0
Axonal degeneration	0	0	1 (1.0)	4* (1.8)	0	0	2 (2.0)	4* (2.3)
Nerve fibre degeneration	2 (1.0)	3 (1.0)	2 (1.0)	0	0	2 (1.0)	3 (1.0)	0
Tissues examined	4	4	4	4	4	4	4	4

( ) = average severity of animals with lesion: 1 (minimal) to 5 (severe)

* = Significantly different from control (p≤0.05)

CONCLUSION

In this subchronic oral gavage study in the dog, compound-related effects were: 1) seizures in the 30 mg/kg dose group males and females and 2) morphologic changes characterized as axonal degeneration noted particularly within the sensory tract of the dorsal spinal cord, with much less involvement of the sciatic nerve and brain stem. These lesions were present in both sexes of the 30 mg/kg dose group with some affected animals in the 15 mg/kg dose group. The LOAEL for this study is 15 mg/kg/day. The NOAEL for this study is 7.5 mg/kg/day. There were no study deficiencies.