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: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

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:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan 12 Nousan No 8147 (2001)

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:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: males 8 weeks, females 11 weeks
- Weight at study initiation: Mean initial weights at study start: males 229 g, females 203 g
- Fasting period before study: no, dermal study
- Housing: Individually into Makrolon cages Type Illhon lowdust wood granules (supplier: Ssniff Spezialdiaeten Inc. Soest/Westfalen, Germany; manufacturer: J. Rettenmeier, Ellwangen-Holzmuehle, Germany). Cages and bedding were replaced weekly by new ones. For environmental enrichment wooden blocks supplied by Papvei OY, 73620 Kortteinen, Finland were provided to each cage.
- Diet (e.g. ad libitum): Fixed-formula standard diet (Article No.: 3883.0.15 (pellet) supplied by Provimi Kliba SA, CH-4303 Kaiseraugst, Switzerland) during the acclimatization period and throughout the study.
- Water (e.g. ad libitum): Tap water during the acclimatization period and throughout the study, ad libitum.
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: On the shaved back .
- % coverage: The size of the gauze-layer was the same as the application area, i.e. 30.0 cm². Thus, the application area was greater than 10% of the body surface area of the rat. Depending on the body weight of the animals and the surface area of the gauze covered by the test item layer were (40 mg/kg: 2.25-4.0 cm², 200 mg/kg: 14.0 cm², 1000 mg/kg: 27.5-30.0 cm² in males, and 40 mg/kg: 2.25 cm², 200 mg/kg: 12.25 cm², 1000 mg/kg: 24.75-27.5 cm² in females) the mean amounts of test item applied per cm² skin in each dose group varied only slightly over the application period of 4 weeks within the groups (males 40 mg/kg: 3.0-4.5 mg/cm², 200 mg/kg: 3.3-4.4 mg/cm², 1000 mg/kg: 8.5-10.5 mg/cm²; females 40 mg/kg: 3.6-3.8 mg/cm², 200 mg/kg: 3.3-3.4 mg/cm², 1000 mg/kg: 8.1-8.8 gm/cm²).
- Type of wrap if used: Moist gauze-layer (6.0 x 5.0 cm = 30.0 cm²; moistened with tap water) of a "Cutiplast steril" patch. The gauze-layer was secured in place using "Peha-Haft" cohesive stretch tape (approx. 8 x 23 cm). Additionally, the mobility of the rats was limited by a "Lomir Biomedical Inc." rat jacket.
- Time intervals for shavings or clipplings: One day before the start of the treatment the back and flanks of the rats were shorn. Any regrowth of hair was shaved off the skin areas marked for treatment twice weekly.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, treated area cleaned with soap and water
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 40, 200, 1000 mg/kg
- Constant volume or concentration used: yes, always neat test substance applied
- For solids, paste formed: no, dry test material was applied to moistened gauze pads

VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable, only used for moistening of gauze pads

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, semiocclusive dressing

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analytical determinations (stability, homogeneity) were not performed, because the test item was applied neat and was only moistened with tap water immediately before application. For the treatment the purity was not taken into account.

Duration of treatment / exposure:

29/30 days (males/females; males 22 applications, females 23 applications)

Frequency of treatment:

6h/day, 5 days/week (week 1-3) and daily thereafter for at least the final 8 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day

Remarks:

nominal per unit body weight

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Remarks:

nominal per unit body weight

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

nominal per unit body weight

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected on the basis of the results of a pilot study. In this study (T7076734) 2 males and 2 females per dose group were treated with doses of 100, 500 and 1000 mg/kg by dermal application. No treatment-related clinical findings were recorded. Body weight development was roughly comparable up to 1000 mg/kg to that of the corresponding control group. The assessment of skin reddening and measurement of skinfold thickness gave no treatment-related effects. Determination of weights of liver, kidneys and testes gave no evidence for treatment-related effects. The weights of spleen seemed to be slightly lower at 1000 mg/kg. Based on these results the dose scheme of 40, 200 and 1000 mg/kg bw/day was chosen.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for morbidity and mortality, once daily on weekends and public holidays; clinical examinations daily.
- Cage side observations included: any clinical signs and abnormalities, assessment of body surfaces and orifices, posture, general behaviour, breathing and excretory products.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, including open field observations in a standard arena for behavioural observations.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Shaved skin areas were examined for skin reddening before start of the study and each day of treatment during the study; swelling was assessed before application and on days 1, 4, 8, 11, 15, 18, 22, 25 and 29 by measuring skinfold thickness on the back in the center of the treatment area using a cutimeter or skinfold caliper by Kroeplin. Measurements were taken at two different locations within the treatment area and means were calculated.
The assessment of reddening was done in conformance with guidelines published by the US Dept. of Agriculture (US-Federal Register, 38, 187 27019, 1973) and according to DRAIZE (The Appraisal of Chemicals in Food, Drugs and Cosmetics, 26-30. Association of Food and Drug Officials of the United States, Austin, Texas, 1959).

BODY WEIGHT: Yes, body weight and body weight gains.
- Time schedule for examinations: Before first treatment and then weekly thereafter. Furthermore, body weights were recorded immediately before scheduled necropsy for calculation of relative organ weights.

FOOD CONSUMPTION: Yes, weekly on days 8, 15, 22 and 29.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day and g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly on days 8, 15, 22 and 29. Water consumption for each animal determined and mean daily water consumption calculated as g water/animal/day and g water/kg body weight/day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before start of treatment and near the end of the treatment
- Dose groups that were examined: All animals were subjected to inspection before start of the study, control and high dose animals were again tested near the end of the treatment period.

The pupillary reflex of both eyes was first tested in a darkened room and the anterior regions of the eye were inspected. After dilating the pupils with Mydriaticum Stulln drops, the refractive elements of the eye as well as iris and fundus were examined using an indirect ophthalmoscope. In addition, the optical media were examined with a ZEISS photo-slit lamp.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning from the retro-orbital venous plexus on day 28 (males) and on day 29 (females).
- Anaesthetic used for blood collection: Yes carbon dioxide anesthesia (80% CO2/20% O2) was used for collection of blood from the retro-orbital plexus.
- Animals fasted: No
- How many animals: All animals per group.
- Parameters examined: Differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, hemoglobin concentration, hematocrit, leukocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning from the retro-orbital venous plexus on day 28 (males) and on day 29 (females) under carbon dioxide anesthesia. Blood samples for determination of glucose were taken from the caudal vein of non-anesthetised animals.
- Animals fasted: No
- How many animals: All animals per group.
- Parameters examined: Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, total bilirubin, cholesterol, creatinine, total protein, triglycerides, urea, glucose, chloride, calcium, inorganic phosphate, potassium, sodium.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals scheduled for necropsy were sacrificed by exsanguination under deep diethyl ether anesthesia. Necropsy was a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Organs and tissues or representative pieces of them were fixed in 10% neutral buffered formalin or Davidson's solution. The following organs and tissues were analysed: Abnormalities, Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Esophagus, Eyes, Eyelids, Exorbital lacrimal glands, Femur (with joint), Harderian glands, Head (with skull cap, Nasal cavity), Nasal Cavity, Heart, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Peyer's patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mandibular, mesenteric, popliteal), Optic nerves, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulating glands), Skeletal muscle (thigh), Skin (mammary region), Skin application site (treated and untreated), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow, Stomach, Testes, Thymus, Thyroid glands (with parathyroids), Tongue, Trachea, Ureters, Urethra, Urinary bladder, Uterus (with cervix), Vagina, Zymbal's glands, Physical identifier.

Changes were described in terms of localization, size, color and consistency whenever appropriate.


HISTOPATHOLOGY: Yes, the following organs and tissues of animals of generally the control and the high dose group were examined histopathologically: Abnormalities (all groups), Adrenal glands, Aorta, Brain (cerebrum, cerebellum, brain stem), Epididymides, Esophagus, Eyes, Femur (with joint), Heart, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mandibular, mesenteric), Optic nerves, Ovaries, Oviducts, Pancreas, Pharynx, Pituitary gland, Prostate, Salivary glands (parotid, submandibular, sublingual), Sciatic nerve, Seminal vesicles (incl. coagulating glands), Skin (mammary region), Skin application site (treated and untreated), Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with Bone Marrow, Stomach, Testes, Thymus, Thyroid glands (with parathyroids), Trachea, Urinary bladder, Uterus (with cervix).

Other examinations:

ORGAN WEIGHT:
The following organs of the animals sacrificed at the end of the treatment were weighed before fixation: Brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), ovaries, uterus, epididymides and testes (both).

Statistics:

For the statistical evaluation of samples drawn from continuously distributed random variables, three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests) where appropriate.

Statistical evaluations on body and organ weight data as well as food/water intake were done using the Dunnett-test in connection with a variance analysis. Evaluating clinico-chemical parameters an analyses of variance followed by a Dunnett test, an adjusted Welch test or a Kruskal-Wallis test was performed. For all these tests SAS® routines were used.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

not test substance-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

not test substance-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

not test substance-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

not test substance-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until the scheduled end of the study.

Skin reddening and incrustation were observed in five females at 40 mg/kg, seven females at 200 mg/kg and in seven females at 1000 mg/kg. However, as this was observed only on a few days (on day 18 and 19 for one female of the 1000 mg/kg group and maximally between day 3 and 7 of the study for the other animals) this is not regarded to be a test substance-related effect, but a consequence of the technical application process.
No effect on the mean skinfold thickness was observed up to 1000 mg/kg in both sexes.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related effect on body weight development was observed up to 1000 mg/kg.

FOOD CONSUMPTION
The food intake in the treated groups was comparable to that of the respective control group.

WATER CONSUMPTION
The water intake in the treated groups did not relevantly differ from that of the respective control group.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmological examinations of the control and of the high-dose group animals performed near the end of the study period afforded no evidence of treatment-related effects.

HAEMATOLOGY
No treatment-related effects were observed on parameters of red and white blood cells, as well as on platelet counts and coagulation time up to 1000 mg/kg in both sexes. A few values, which were significantly different from control values are considered to be of no toxicological relevance since either the differences from control were slight and/or they did not show a correlation with the dose administered.

CLINICAL CHEMISTRY
The investigations gave no evidence for treatment-related effects on activities of enzymes or concentrations of substrates and electrolytes in peripheral blood of males and females up to 1000 mg/kg. A few values which were significantly different from control values are considered to be of no toxicological relevance since either the differences from control were negligibly low or they did not show a correlation with the dose administered and with the time of treatment.

ORGAN WEIGHTS
Determination of organ weight resulted in no toxicologically relevant differences between treated groups and the respective control group.

GROSS PATHOLOGY
There was no evidence of any treatment-related gross pathological finding. The necropsy findings were randomly distributed throughout the groups and are known to occur frequently in animals of this species and age in the test laboratory.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no evidence of any histopathological finding that had to be attributed to dosing with the test compound.
The investigation of the skin at the application site revealed in some animals minimal mononuclear infiltrates below the epidermis or a minimally increased thickness of the epidermal layers (epidermal hyperplasia) of the treated or the untreated skin. These findings were unrelated to treatment when comparing the control group to the treatment group and the treated and the untreated skin of an individual animal. They are therefore regarded to be minimal sequels of the technical application process. The formulation was thus locally well-tolerated and there was no evidence of any systemic effect up to and including 1000 mg/kg bw/day.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest dose tested

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest dose tested

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

CONCLUSION

Gross and histopathological investigations revealed that the formulation was locally well tolerated up to and including 1000 mg/kg bw/day and there was no evidence of any systemic effect up to and including 1000 mg/kg bw/day. In summary, 1000 mg/kg bw/day was determined as the No-Observed-Adverse-Effect Level for the local skin effects in both sexes. The overall No-Observed-Adverse-Effect Level under the conditions described was 1000 mg/kg bw/day for both males and females.