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REACH

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

EC number: 936-023-6 | CAS number: 950782-86-2

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

OECD 409 subchronic, dog, gavage: NOAEL = 7.5 mg/kg bw/day; LOAEL = 15 mg/kg bw/day: STOT RE 2 (neurotoxicity) 
OECD 452 chronic (1y), dog, feed: NOAELmale/female = 2 mg/kg/day; LOAELmale/female = 6/7 mg/kg/day: STOT RE 2 (neurotoxicity) 
OECD 453 combined chronic/carcinogenicity (2y), rat, feed: NOAELmale/female = 12/17 mg/kg bw/day; LOAELmale/female = 118/167 mg/kg bw/day 
OECD 408 subchronic, rat, feed: NOAELmale/female = 338/410 mg/kg bw/day; LOAELmale/female = 689/809 mg/kg bw/day 
OECD 410 subacute, rat, dermal: NOAEL ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference 1

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.
Qualifier:: according to guideline
Guideline:: OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
Deviations:: no
GLP compliance:: yes
Limit test:: no
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
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
Dose / conc.:: 7.5 mg/kg bw/day (actual dose received)
Dose / conc.:: 15 mg/kg bw/day (actual dose received)
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
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.
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.
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
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
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

Reference 2

Endpoint:: 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.
Qualifier:: according to guideline
Guideline:: OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.4100 (Chronic Toxicity)
Deviations:: no
GLP compliance:: yes
Limit test:: no
Species:: dog
Strain:: Beagle
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Marshal BioResources USA, Inc, North Rose, New York
- Age at study initiation: Males: 7-9 months; Females: 7-9 months
- Weight at study initiation: Males: 6.9-10.1 kg; Females: 6.0-7.8 kg
- Fasting period before study: not applicable
- Housing: Individually housed in stainless steel runs.
- Diet: Purina Mills Certified Dog Diet ETTS 5006-3 available for ad libitum consumption, except when animals were fasted prior to bleeding.
- Water: Tap water provided continuously for ad libitum consumption.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-29
- Humidity (%): 30-70
- Air changes (per hr): Averaged at least 13.43
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 June 2006 To: 22 June 2007
Route of administration:: oral: feed
Vehicle:: other: corn oil (1% of diet) with ethanol
Details on oral exposure:: DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Mills Certified Dog Diet ETTS 5006-3.
- Storage temperature of food: Freezer conditions until presented to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil, at 1% by weight of the diet, along with ethanol, was used as a vehicle to dissolve and suspend the test substance prior to being mixed in the diet. The control diet was prepared the same way as the treated diet, excluding only the test substance.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The concentration of the active ingredient in the feed was verified for the first three weeks of treatment, then once every 4 or 5 weeks until the end of the study. The mean concentrations for the study were 96 to 97% of the nominal levels; %RSD values ranged from 5.8 to 6.4.
Duration of treatment / exposure:: 368-372 days
Frequency of treatment:: daily
Dose / conc.:: 60 ppm
Remarks:: corresponding to 2 mg/kg bw/day for males and females
Dose / conc.:: 225 ppm
Remarks:: corresponding to 6 mg/kg bw/day for males and 7 mg/kg bw/day for females
Dose / conc.:: 450 ppm
Remarks:: corresponding to 12 mg/kg bw/day for males and 11 mg/kg bw/day for females
No. of animals per sex per dose:: 4
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: The nominal concentrations chosen for this study were 0 (concurrent vehicle control) 60, 225, and 450 ppm (4/sex/dose) of technical grade test substance mixed with dog ration. Selection of these doses was based on a 90-day oral gavage study in the Beagle dog in which seizures were observed at a dose of 30 mg/kg bw/day, but not at 15 mg/kg bw/day.
Positive control:: none
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 of toxicity.

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

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; also immediately prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, daily throughout the study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once pre-exposure and just prior to necropsy.
- Dose groups that were examined: All animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Once pre-exposure and during weeks 13, 26, 39 and 52.
- Anaesthetic used for blood collection: No, blood taken via jugular venipuncture.
- Animals fasted: Yes, overnight
- How many animals: All animals.
- Parameters examined: Hematocrit, hemoglobin, leukocyte count (WBC), erythrocyte count (RBC), platelet count, thromboplastin time, prothrombin time, leukocyte differential count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, blood cell morphology, red blood cell distribution width (RDW), hemoglobin distribution width (HDW).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once pre-exposure and during weeks 13, 26, 39 and 52.
- Animals fasted: Yes, overnight
- How many animals: All animals.
- Parameters examined: Calcium, chloride, phosphorus, potassium, sodium, alkaline phosphatase (ALK), creatine phosphokinase (CK), lactic acid dehydrogenase (LD), alanine aminotransferase (ALT/SGPT), aspartate aminotransferase (AST/SGOT), gamma glutamyl transferase (GGT), Albumin (ALB), creatinine (Creat), urea nitrogen (Urea-N), 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: Once pre-exposure and during weeks 13, 26, 39 and 52
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- 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).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During study months 7, 8, 9, 10, 11 and pre-termination.
- Dose groups that were examined: All animals.
- Battery of functions tested: Signs of neurotoxicity, body temperature.
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. Tissues collected: 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 tissues collected (except vagina and physical identifier) were processed, embedded in paraffin, sectioned, mounted, and stained with hematoxylin and eosin (H&E) for examination under the light microscope. Histopathologic evaluation was conducted on all protocol-required tissues from the control and high-dose group animals and on tissues from the low- and mid-dose groups for which compound-related lesions were observed in the high-dose 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.
Clinical signs:: no effects observed
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):: not examined
Ophthalmological findings:: no effects observed
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: no effects observed
Behaviour (functional findings):: no effects observed
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):: adverse
Histopathological findings: neoplastic:: not examined
Details on results:: CLINICAL SIGNS AND MORTALITY
There were no compound-related deaths in males or females at any dietary level. One high-dose group male was sacrificed in-extremis on study day 190 due to seizure.

There were no compound-related clinical findings. The sacrificed high-dose group dog had convulsions/seizures and associated salivation on Study Day 190. Although it has been shown in prior studies that the test substance causes seizures, it cannot be determined if the seizures in this animal were due to the test compound or bilateral dilated cerebral ventricles observed at necropsy. However, as seizures were only observed in 1 of 8 high-dose group animals and as seizures were not observed in previous studies at the same dose with the test compound administered as a bolus dose by oral gavage, it is considered that the seizure in this single high-dose group dog was not caused by the test substance.

BODY WEIGHT AND WEIGHT GAIN
There was no compound-related effect on body weight in males or females at any dietary level. Body weights for high-dose group males were non-statistically lower than the control group throughout the study, but this is considered to be due to the low body weight for the male high-dose group at the initiation of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no compound-related effects on food consumption in males and females at any dietary level. Sporadic statistically significant differences in food consumption for treated groups as compared to the control group were considered incidental.

OPHTHALMOSCOPIC EXAMINATION
No ocular abnormalities were observed in males or females at any dietary level.

HAEMATOLOGY
There were no compound-related hematology findings in males or females at any dietary level. There were a few statistical or non-statistical fluctuations in the absolute or relative lymphocytes or neutrophils, particularly in the high-dose group, which were more notable in males at several collection intervals. However, all values were within the historical control ranges and were without biological significance.

CLINICAL CHEMISTRY
There were no compound-related clinical chemistry findings in males or females at any dietary level. There were numerous statistical notations, however, they were often similar to pre-study values or due to lower control values. Additionally, they were not biologically changed over multiple bleeding intervals, were not affected in a linear dose fashion, and were within historical control ranges.

URINALYSIS
There were no compound-related urinalysis findings in males or females at any dietary level. In high-dose group males, there was a non-statistical tendency for decreased urine volume and increased urine specific gravity, but there were no other parameter changes, no statistically significant differences, and no associated micropathology findings. In females, urine pH was sometimes statistically decreased, however, this difference from the control group was not dose-related, was similar to pre-study ranges, and did not persist to study termination.

NEUROBEHAVIOUR
No neurological observations were observed by neurological examination at any dietary level.

ORGAN WEIGHTS
There were no compound-related changes in the terminal-body or organ weights in males or females at any dietary level. There were also no statistical differences in the male and female absolute organ weights at any dietary level. There was a statistical decrease in the low-dose group female relative brain weights. There was good correlation between the increased ovary and uterine weights in some females which were indicative of the different stages of the female estrus cycle. Missing weights for one high-dose female and one control male did not affect study interpretation.

GROSS PATHOLOGY
There were no test-compound related gross lesions observed at study termination in either sex. Incidental observations: The male euthanized at study day 190 had grossly observed dilatation of the lateral ventricles in the brain and a pituitary cyst. A pituitary cyst was also seen in one low-dose male and one high-dose female.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test-compound-related lesions were principally observed within the dorsal funiculi (primarily the funiculus cuneatus), a sensory fiber white tract(s) of the spinal cord, in mid- and high-dose males and females. The lesion was characterised by individual nerve fiber changes, coded as axonal degeneration, consisting of fragmented and lysed axonal fibers, sometimes with associated phagocytic macrophages (gitterzellen) forming a digestion chamber. Secondary subtle myelin change (demyelination) was noted. The lesion represents an enhancement of a background change of individual nerve fiber degeneration seen in control animals in a variety of neural tissues, including the spinal cord, brain, and sciatic (peripheral) nerve. Thus, the spinal cord lesion (primary target tissue) was given a semi-quantitative grading scheme whereby greater than 10 individual nerve fibers undergoing a focus of axonal change was grade 1, 15-25 affected fibers was grade 2, and 30 and above affected fibers was grade 3. In both males and females degenerations of grade 1 to 3 (minimal to moderate), significantly different from control in high-dose females, were observed.

The extent of background or test-substance-related change in the brain stem and/or sciatic nerve was much less prominent and, in-fact, subtle. The grade 1 axonal change in the denoted brain sections of high-dose group males was principally with the brain stem most notably associated with the cranial nerve roots adjacent to the paraflocculus lobe of the cerebellum. The extent of the lesion in the brain stem was not comparable to a grade 1 lesion in the spinal cord and only represents 2-4 individual nerve fibers altered in the section(s) evaluated. Likewise, the sciatic nerve lesion was most subtle and represented 2-4 altered individual nerve fibers, which typically would be considered as background incidence if there had not been prominent spinal cord changes.

Non-test-substance-related lesions:
Incidental background lesions were seen in both sexes. Some morphologic changes included C-cell hyperplasia in the thyroid, pituitary cysts, ductular remnants in the thymus, prostatic inflammation, cytoplasmic change in the parathyroid (syncytial cell change), and subtle inflammatory cell infiltrate/inflammation in the lung. The high-dose male euthanised with clinical signs (convulsions/seizures/salivation), was noted grossly to have dilated lateral ventricles in the brain. Microscopic examination and re-evaluation of the wet tissue confirmed the minimal to slight dilatation of the lateral ventricles. However, this finding was interpreted as an incidental finding, occasionally seen in the Beagle dog, and unrelated to test-substance administration. No other animals in this study with test substance-related morphological changes had clinical signs or dilation of the lateral ventricles.
Dose descriptor:: NOAEL
Effect level:: 60 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: other: corresponding to 2 mg/kg bw/day for males and females.
Dose descriptor:: LOAEL
Effect level:: 225 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: histopathology: non-neoplastic
Remarks on result:: other: corresponding to 6 mg/kg bw/day for males and 7 mg/kg bw/day for females.
Critical effects observed:: yes
Lowest effective dose / conc.:: 225 ppm
System:: central nervous system
Organ:: spinal cord
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 2 mg/kg bw/day
Study duration:: chronic
Species:: dog
Quality of whole database:: The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.
System:: central nervous system
Organ:: spinal cord

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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.
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:: no
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
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
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
Dose / conc.:: 40 mg/kg bw/day
Remarks:: nominal per unit body weight
Dose / conc.:: 200 mg/kg bw/day
Remarks:: nominal per unit body weight
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
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.
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.
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
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
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

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.
Qualifier:: according to guideline
Guideline:: OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:: no
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
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
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
Dose / conc.:: 40 mg/kg bw/day
Remarks:: nominal per unit body weight
Dose / conc.:: 200 mg/kg bw/day
Remarks:: nominal per unit body weight
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
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.
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.
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
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
Critical effects observed:: no

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Study duration:: subacute
Species:: rat
Quality of whole database:: The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Oral:

There are three subchronic studies available addressing repeated dose toxicity via the oral route, conducted in rats, dogs, and mice. In the subchronic study in rats, which was performed according to OECD guideline 408, the rats received doses of 200, 5000 and 10000 ppm of the test substance, corresponding to 14, 338, and 689 mg/kg bw/day for males and 16, 410 and 806 mg/kg bw/day for females, for a period of 90 days in their feed. No adverse effects were observed at the dose level of 5000 ppm in either sex. Therefore, this dose level (corresponding to 338 mg/kg bw/day for males and to 410 mg/kg bw/day for females) was determined to be the NOAEL. However, slight effects which were not considered to be adverse, like changes in clinical chemistry, minimal hypertrophy of thyroid follicular cells and increase in TSH levels in males, and enlarged livers in combination with higher liver weights, were observed at this dose level. All of these observations reflect adaptation to test substance exposure by activation of the detoxification metabolism; therefore, this dose level of 5000 ppm is considered as LOEL, as well. At the highest dose level of 10000 ppm, corresponding to 689 mg/kg bw/day in males and 806 mg/kg bw/day in females, one female had to be sacrificed in moribund condition on day 20 after loss of weight and reduced food consumption in the first 2 weeks of the study. Clinical signs in this animal comprised piloerection and hunched posture. Macroscopic examination revealed red foci on the stomach, and although microscopic examination revealed no clear cause of moribundity, the condition of this animal was considered related to treatment. Males demonstrated minimal to moderate hypertrophy of follicular cells in the thyroid gland, which were considered treatment-related and toxicologically relevant, and a statistically significant increase in TSH levels in week 3, which could account for the increase in cell size. Therefore, the LOAEL was determined to be 689 mg/kg bw/day.

However, the subchronic study in dogs, which were dosed with 7.5, 15 and 30 mg/kg bw/day by oral gavage over a period of 90 days, revealed a much higher sensitivity of this species for the adverse effects of the test substance. Clinical signs observed in the high dose group were so severe (seizures in 1/4 males and 2/4 females) that the animals had to be euthanised in extremis. Due to the seizures dosing of the high-dose group was stopped on day 35 and all animals were euthanised on day 36. At the highest dose axonal degeneration and demyelination with phagocytosis of fragmented axonal and myelin debris by macrophages (producing so-called digestion chambers) were observed, which were also found in mid-dose animals at 15 mg/kg bw/day, therefore this dose was determined to be the LOAEL of this study. Accordingly, this study was chosen as a key studies for classification. No adverse effects were observed at 7.5 mg/kg bw/day, which was considered to be the NOAEL. Based on the observation of neuronal degeneration a classification for Specific Target Organ Toxicity after Repeated Exposure (STOT RE) is suggested.

Additionally, there is a subchronic toxicity study in mice available, in which the animals received doses of 100, 500 and 1200 ppm, corresponding to 19, 91 and 218 mg/kg bw/day for males and 23, 118 and 256 mg/kg bw/day for females, for 91 to 93 days in their feed. In this study no effects were observed at the dose level of 500 ppm. Only slight, non-adverse effects on body weight, food consumption and clinical chemistry, and some minor histopathological changes, which were not considered toxicologically relevant, were observed at a dose level of 1200 ppm (LOEL). Therefore, this dose of 218 mg/kg bw/day in males was also considered as NOAEL in this study. As testing was not performed at doses high enough to cause significant toxicological effects, this study was considered less relevant for assessment.

Two chronic studies are available addressing toxicity after repeated exposure, the first one a chronic toxicity study according to OECD guideline 452, performed over 1 year in dogs, the second one a combined chronic toxicity and carcinogenicity study according to OECD guideline 453, performed over 2 years in rats.

Comparable to the subchronic studies the dog proved to be the most sensitive species after chronic exposure, as well. Accordingly, this study was selected as second key study. In this study the animals were exposed to dose levels of 60, 225 and 450 ppm in the feed, corresponding to 2, 6 and 12 mg/kg bw/day for males and 2, 7, and 11 mg/kg bw/day for females, for a period of 368 to 372 days. No treatment-related effects were observed at the dose level of 60 ppm. Therefore, this dose level was determined to be the NOAEL in this study, corresponding to 2 mg/kg bw/day for both males and females. This NOAEL also served as basis for the derivation of the DNEL. Beginning at the mid-dose level of 225 ppm, corresponding to 6 and 7 mg/kg bw for males and females, respectively, minimal to moderate axonal degeneration of the dorsal funiculi was observed in the histopathological examination. These lesions consisted of fragmented and lysed axonal fibers, sometimes associated with phagocytic macrophages forming digestion chambers. Therefore, a dose level of 6 mg/kg bw/day was determined as LOAEL. The same type of lesions was also observed in the subchronic study; the classification of the test substance for STOT RE is further supported by the chronic results.

In the combined chronic toxicity and carcinogenicity study in rats the animals were exposed to dose levels of 300, 3000 and 10000/6000 ppm in their diet, corresponding to 12, 118, and 414 mg/kg bw/day for males and 17, 167, and 661/452 mg/kg bw/day for females over a period of 104 weeks. In females the dietary level in the high-dose group had to be lowered from 10000 to 6000 ppm from week 41 on due to marked clinical signs like dilated pupils, limited use of limbs, uncoordinated movements, tremors, splayed hindlimbs, low alertness, soiled anogenital region and nose, piloerection, wasted appearance and laboured, rapid and noisy respiration. In the liver, hepatocellular hypertrophy, macrovacuolation, single cell necrosis and multinucleated hepatocytes were noted. In the kidney, a higher incidence and severity of microscopic intracellular brown pigments was noted in females. In the thyroid gland, higher incidences of follicular cell hypertrophy were observed in males, and colloid alteration in both males and females. In the eye, higher incidences of degeneration like bilateral and peripheral retinal atrophy were noted in females. In the pituitary gland and the brain, degenerative changes concerning the neurohypophysis were observed; higher incidences of pars nervosa and median eminence vacuolation were noted in the pituitary gland and the brain, respectively, in both sexes, supporting the observations from the repeated dose studies in dogs. The incidence of clinical signs clearly decreased after lowering the dose level to 6000 ppm. At the mid-dose of 3000 ppm, corresponding to 118 and 167 mg/kg bw/day in males and females, respectively, clinical and histopathological findings comparable to the high dose level, although less pronounced, were observed. Therefore a dose level of 118 mg/kg bw/day was determined as chronic LOAEL for rats. No effects were observed at the low-dose of 300 ppm (corresponding to 12 and 17 mg/kg bw/day in males and females, respectively); a dose level of 12 mg/kg bw/day was determined as chronic NOAEL for rats.

Both rats and dogs demonstrate neurodegenerative effects after repeated test substance administration via the oral route, although only observed after 2 years in the rat. In comparison, the dog is more sensitive, as these effects already occured at lower dose levels and after shorter exposure periods. Therefore, the subchronic dog study was chosen as basis for classification purposes, although the dog is not the preferred species, and threshold levels are defined for rats; due to the interspecies differences between rats and dogs these threshold levels are difficult to apply for dogs, as they are known to be very sensitive for neuronal changes, anyway. Based on the observed effect levels in rats, a classification for Specific Target Organ Toxicity after Repeated Exposure would not be required. Accordingly, this approach can be considered as rather conservative. For the derivation of the DNEL the NOAEL from the chronic study in dogs was chosen as starting point. Therefore, this approach might result in a very low DNEL and an overestimation of the risk, but ensures an additional safety margin for human exposure.

Dermal:

In addition to the studies via the oral route a subacute dermal study in rats is available. In this study the animals were exposed for 4 weeks to doses of 40, 200 and 1000 mg/kg bw/day of the test substance. No effects of toxicological relevance were observed in this study up to the highest dose level of 1000 mg/kg bw/day, therefore this dose level was considered to be the NOAEL for systemic and local effects.

Inhalation:

According to Regulation (EC) No 1907/2006, 8.6.2, column 2, administration by inhalation is not required. Due to a low vapour pressure inhalation is no relevant route of exposure, and extensive results from subchronic oral studies for three species (rat, mouse, dog) and chronic oral studies in two species (rat, dog) are available. Furthermore, results from a subacute dermal study in rats were reported. Acute toxicity studies demonstrate that toxicity via the oral route is more relevant for assessment, although particles of inhalable size are present. However, appropriate RMMs are in place, and additional inhalation testing would neither improve risk assessment nor safety of applications.

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008 the test substance has to be classified as causing Specific Target Organ Toxicity after Repeated Exposure (STOT RE) due to the neuronal lesions observed in dogs after subchronic and chronic exposure, and in rats after chronic exposure.

CLP: STOT RE 2; H373: May cause damage to organs through prolonged or repeated exposure.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

	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

Registration Dossier

Registration Dossier

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Diss Factsheets

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

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

Referenceopen all close all