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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Oral subchronic toxicity

key; M-027268-01-1; subchronic (90 d, rat): NOAEL (oral) 27.9 mg/kg bw/day (males) and 34.0 mg/kg bw/day (females)

key; M-036499-02-1; subchronic (90 d, dog): NOAEL (oral) 19.3 mg/kg bw/day (males) and 21.2 mg/kg bw/day (females)

key; M-036542-01-1; subchronic (1 y, dog): NOAEL (oral) 36.3 mg/kg bw/day (males) and 40.1 mg/kg bw/day (females)

 

Oral chronic toxicity

key; M-031986-05-1; chronic/carcinogenicity (2 y, rat): NOAEL (chronic, oral) 27.4 mg/kg bw/day (males) and 9.7 mg/kg bw/day (females)

key; M-032363-03-1; carcinogenicity (78 weeks, mouse): NOAEL (chronic, oral) 47.2 mg/kg bw/day (males) and 65.1 mg/kg bw/day (females)

 

Dermal subacute toxicity

key; M-027480-01-1; subacute (4 weeks, rat): NOAEL (dermal) 1000 mg/kg bw/day (females, males)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - Aug 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
housing conditions were slightly different to the guideline, no detailed clinical observations (sensory reactivity), blood clotting time, weight of prostate and pituitary gland not provided, justification for choice of vehicle not given
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
assumed to be 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen since it is the recommended rodent species in OECD TG 408.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 258 - 304 g for males and 176 - 211 g for females
- Housing: individually housed in stainless steel wire-mesh cages with deotized cage board in the bedding tray
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
Feed, water, and corn oil (used in diet preparation to facilitate mixing of the test substance in the feed) were periodically sampled and analyzed for a variety of potential impurities. The results of these analyses, which have been archived, were unremarkable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
other: acetone/corn oil mixture
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Replacement admixtures for each treatment group were prepared weekly
- Mixing appropriate amounts with (Type of food): acetone/corn oil mixture was used as a vehicle to suspend the test substance prior to mixing in the basal diet
- Storage temperature of food: stored under refrigerator conditions

VEHICLE
- Purity: Corn oil was periodically sampled and analyzed for a variety of potential impurities. The results of these analyses, which have been archived, were unremarkable.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration:
The concentration of the test substance in the various test diets was analytically verified 4 separate times during the in-life phase of this study (weeks 1, 5, 9, and 14). The mean analytical concentrations for each dose group were 138, 423, and 2945 ppm, with all values remaining within approximately 15% of the corresponding nominal concentrations of 150, 500, and 3000 ppm, respectively.

Homogeneity:
Feed was analyzed for uniformity of distribution/homogeneity of the test substance in rat diet. Therefore, three samples were taken from each of three distinct layers (top, middle and buttom) in a mixing bowl containing a nominal concentration of either 50 or 5000 ppm. The mean concentration was determined to be 46.5 ppm and 4341 ppm, respectively. Based on the results, it was judged that the test substance was homogeneously distributed in the feed over a concentration range of 50 - 5000 ppm (with a coefficient of variation below to 10%).

Stability:
A time and temperature analysis of feed containing nominal concentrations of 50 and 5000 ppm of the test substance was conducted. Based on the criteria of at least 80% recovery of the initial chemical concentration, the test substance mixed in rodent ration was judged to be stable following room temperature storage for a minimum of 7 days and refrigerator storage for a minimum 28 days, respectively, over a concentration range of 50 - 5000 ppm.
Duration of treatment / exposure:
approximately 14 weeks
Frequency of treatment:
continuously in the feed
Dose / conc.:
150 ppm
Remarks:
corresponding to 9 mg/kg bw/day (males) and 10.9 mg/kg bw/day (females)
Dose / conc.:
500 ppm
Remarks:
corresponding to 27.9 mg/kg bw/day (males) and 34.0 mg/kg bw/day (females)
Dose / conc.:
3 000 ppm
Remarks:
corresponding to 202 mg/kg bw/day (males) and 254.2 mg/kg bw/day (females)
No. of animals per sex per dose:
Main groups: 15
Recovery groups: 15 (control and high-dose only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selection was based on a previous 13-week oral feeding study with the test substance.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Post-exposure recovery period in satellite groups: 7 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once each week on all animals

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and after exposure duration
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to their respective termination
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: platelet count (PLTS), leucocyte count (WBC), erythrocyte count (RBC), hemoglobin (Hgb, Hg), hematocrit (HCT, PCV), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), leukocyte differential counts, erythrocyte morphology, prothrombin time (PT), reticulocyte (Retic) and heinz body (HZ) counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to their respective termination
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: sodium (Na, Na+), potassium (K, K+), chloride (Cl), magnesium (MG), urea nitrogen (UN), glucose-fasting (Glue), creatinine (Creat, Crea), uric acid (Uric-A), triglyceride (Trig), cholesterol (Choi), creatine kinase (CK, CPK), aspartate aminotransferase (AST, GOT, SGOT), alanine aminotransferase (ALT, GPT, SGPT), gamma-glutamyl transpeptidase (GGT, GGTP), alkaline phosphatase (ALP), total bilirubin (T-Bili), direct bilirubin (D-Bili), total protein (T-Prot, Prot), albumin (ALB, ALBU), phosphorus (Phos, PO4), calcium (Calc, Ca), and globulin (Glob), Thyroxine (T4), Triiodothyronine (T3), and Thyroid stimulating hormone (TSH)
Enzyme activities not specified in the guideline: cytochrome P-450 (Cyto P-450), N-demethylase (N-Demeth), O-demethylase (O-Demeth), Ethoxyresorufin O-deethylase (EROD), and Pentoxyresorufin O-dealkylase (PROD) which are all hepatic enzymes and lactate dehydrogenase (LD, LDH, LDH-L) which indicates tissue damage

PLASMA/SERUM HORMONES/LIPIDS: refer to the list above under clinical chemistry

URINALYSIS: Yes
- Time schedule for collection of urine: the week prior to blood collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: pH (pH), urine protein (Pro), urine glucose (Glu), ketones (Ket), urine bilirubin (Bil), urine blood (Bid), urobilinogen (Uro), Leukocytes (U-Leu), and nitrite (Nit), urine volume (uVOL), urine appearance, urine clarity (Clarity), urine color (Color), specific gravity (Sp.Gr.), and microscopic observation of solids (Micro)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
- Estrous cycle staging: The estrous cycle of each female was determined by examining daily vaginal smears and charted over a 3-week period prior to their respective termination. If possible, animals were selected for terminal sacrifice that were determined to be in the same estrous cycle stage in order to alleviating any possible effects of non-synchronous estrous cycle stage on various measured parameters (uterus weight, follicular population, etc.).
Sacrifice and pathology:
All animals were sacrificed by exsanguination and/or CO2 asphyxiation and subject to a postmortem examination for documenting and saving all gross lesions, weighing designated organs, and collecting representative tissue specimens for histopathologic evaluation. All tissues from control and high dose (3000 ppm) animals, as well as gross lesions from all animals were examined under a light microscope.

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testicles, thymus, thyroid, and uterus

HISTOPATHOLOGY: Yes
Histopathologic evaluation included the following organs/tissues: integumentary- skin, mammary gland; musculoskeletal- bone (femur, rib/cc jet, sternum), joint (fem/tib), muscle (protocol), skull; respiratory- lungs, larynx, trachea, nasopharynx; cardiovascular- heart, aorta; hematopoietic- spleen, bone marrow, lymph node (cervical), lymph node (mesenteric), thymus; digestive- liver, cecum, colon, esophagus, pancreas, rectum, salivary gland, small intestine (duodenum, ileum, jejunum), stomach; urogenital- kidneys, ovaries, testicles, cervix, clitoral gland, epididymis, preputial gland, prostate, seminal vesicles, urinary bladder, uterus, vagina; endocrine- adrenal glands, parathyroid, pituitary, thyroid; nervous- brain (cerebellum, cerebrum-midbrain, medulla/pons), optic nerve, sciatic nerve, spinal cord (cervical, lumbar, thoracic); special senses- exorbital lacrimal gland, eyes, harderian glands, Zymbal's gland; gross lesions, physical identifier, and associated tissues
Statistics:
Continuous data that were examined statistically were evaluated initially for equality or homogeneity of variance using Bartlett's test (Snedecor and Cochran, 1967). Group means were further analyzed by a one-way variance analysis (ANOVA) (Snedecor and Cochran, 1967) followed by Dunnett's test (Dunnett, 1955, 1964). In the event of unequal variances, and at the discretion of the study director, data were subject to non-parametric procedures consisting of a Kruskal-Wallis ANOVA (Hollander and Wolfe, 1973) followed by the Mann-Whitney-U test for between-group comparisons. Frequency data were initially examined for trends; data suggestive of a potential effect were then statistically evaluated using the chi-square, Fisher exact, or chi-square and Fisher exact tests. On a case by case basis, data were subject to additional statistical procedures other than those mentioned above. For the Bartlett test, a probability (p) value < 0.001 was considered significant; for all other statistical tests, differences with p values < 0.05 were considered statistically significant.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs attributable to exposure to the test substance were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival of the treated animals was comparable to control animals. One male rat given 500 ppm was found dead on Day 17. The cause of death was undetermined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight gain was comparable to the control in both sexes at doses up to and including 500 ppm. Animals given 3000 ppm showed a reduced body weight gain for both sexes of approximately 11 and 12% in the male and female, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption and utilization was similar to the control at all doses tested.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Pre- and post-exposure ophthalmological examinations provided no evidence that the test substance induced ophthalmic toxicity in either sex at any dose tested.
Haematological findings:
no effects observed
Description (incidence and severity):
Evaluation of the data from blood collected approximately 14 weeks into the study provided no indication that the test article induced change in either sex at any dose tested.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the highest dose tested, increased hepatic enzyme activities were measured. There was statistically increased induction of N-demethylase and O-demethylase enzyme activity in the liver tissue of the 3000 ppm males. Slight, non-statistical increase in cytochrome P-450 in 3000 ppm males and females associated with increased N-demethylase in 3000 ppm females suggest subtle response to compound exposure.
In general, these changes in enzyme activity may reflect an adaptive response by the liver to an increased need to facilitate the metabolism and excretion of an exogenously administered test substance. Morphological evidence of a direct toxicological effect of the test item on the liver was not observed.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
Evaluation of urine chemistries from samples collected approximately 14 weeks into the study provided no indication that the test substance induced change in either sex at any dose tested.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative organ weight increases were seen for brain, kidney, and testis of 3000 ppm males and for brain and heart of 3000 ppm females at the 13-week termination. Relative organ weight increases were also present in 3000 ppm recovery females for brain, lung, spleen, and ovaries. These variations were considered secondary due to alterations in body weight gain observed in both sexes at the highest dose tested. This conclusion was also supported by the lack of microscopic evidence of direct toxicological effect of the test item on any tissue examined in this study.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross lesions at necropsy.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increased incidence of pigmentation which was associated with exposure to the test item was observed in the spleen of 3000 ppm males (13/15) compared to the control animals (6/15). However, this microscopic lesion was not present at the recovery groups termination. The increased incidence of pigmentation was suggested to be deposition of hemosiderin, an iron-containing pigment derived from the hemoglobin of disintegrating red blood cells.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle staging:
There was no effect of treatment on the duration and stages of the estrous cycle in females at any dose level, and no effects on ovarian follicular populations.
Details on results:
Recovery group:
By conclusion of the recovery period, reversibility was suggested in every parameter that had been influenced; many parameters had fully reverted to control levels.
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponding to 27.9 mg/kg bw/day for males and 34.0 mg/kg bw/day for females
Key result
Dose descriptor:
LOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 202.0 mg/kg bw/day for males and 254.2 mg/kg bw/day for females
Key result
Critical effects observed:
no
Conclusions:
The study was performed under GLP conditions and similar to OECD TG 408. Deviations from the current OECD guideline (2018) were related to detailed clinical observations, specific blood parameters and specific organ weights. However, the study is considered reliable and valid. Under the conditions of this study, the NOAEL for subchronic toxicity was considered to be 500 ppm, corresponding to 27.9 mg/kg bw/day for males and 34.0 mg/kg bw/day for females based on the occurrence of reduced body weight gain in both sexes, and hepatic microsomal enzyme induction in both sexes at 3000 ppm and pigmentation of the spleen in males only at 3000 ppm.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jan 1998 - 14 Mar 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted 1981
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: Covance Research Products, Inc., Virginia, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 5 - 6 months
- Weight at study initiation: 8.5 - 11.4 kg for males and 7.6 - 9.2 kg for females
- Fasting period before study: None
- Housing: individually housed in an elevated, stainless-steel cage measuring approximately 90 x 83 x 79 cm (d x w x h)
- Diet: PMI® Certified Canine Diet® Meal #5007, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
No contaminants were known to be present in the diet or water at levels which might interfere with this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27.6
- Humidity (%): 30.1 - 48.5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Jan 1998 To: 21 Apr 1998
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diets were prepared weekly
- Mixing appropriate amounts with (Type of food): basal diet
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the concentration of the test substance was done by HPLC at the testing facility.

Concentration:
Analyses were performed on one of the duplicate samples collected from the middle of each batch at Weeks 1 and 13. Results of routine concentration analyses indicated that all formulations prepared for Weeks 1 and 13 were within 7.7% of target concentration.

Homogeneity:
Prior to initiation of dosing, homogeneity was investigated on diet formulations prepared at 50, 325 and 2500 ppm. Analyses were performed in duplicate from samples taken from the top, middle, and bottom of each formulation. Analyses indicated that the test material was homogeneously mixed, the relative standard deviation values were 1.10, 1, 13 and 0.81% for the 50, 325, and 2500 ppm concentrations, respectively.

Stability:
Diet formulations at concentrations of 50, 325 and 2500 were analyzed to assess 0-, 7-, 9- and 14-day stability at room temperature. Results of stability analyses indicated that the formulations were stable for 14 days at room temperature.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Dose / conc.:
325 ppm
Remarks:
corresponding to 9.2 mg/kg bw/day (males) and 9.6 mg/kg bw/day (females)
Dose / conc.:
650 ppm
Remarks:
corresponding to 19.3 mg/kg bw/day (males) and 21.2 mg/kg bw/day (females)
Dose / conc.:
1 500 ppm
Remarks:
corresponding to 40.9 mg/kg bw/day (males) and 42.1 mg/kg bw/day (females)
Dose / conc.:
2 250 ppm
Remarks:
corresponding to 58.2 mg/kg bw/day (males) and 61.8 mg/kg bw/day (females)
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Selection of dose was based on a 4-week study in dogs in which a concentration of 1250 ppm had clearly no observable adverse effect, but a concentrations of 2500 ppm and 5000 ppm exceeded the maximum tolerated dose.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: for evidence of toxic or pharmacologic effect and for mortality and moribundity (twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least once prior to treatment, on the first day of treatment, and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during Week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to treatment and during Weeks 5 and 13
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood cell morphology, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment and during Weeks 5 and 13
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, alkaline phosphatase, cholesterol, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, sodium,
potassium and chloride

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: during Weeks 5 and 13
- Metabolism cages used for collection of urine: urine samples were collected overnight in clean containers placed under the drainage opening of each cage
- Animals fasted: Yes, overnight
- Parameters checked: appearance, protein, glucose, specific gravity, volume, pH, ketones, blood microscopic examination of sediment and total bilirubin

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during Weeks 1, 5, and 13
- Dose groups that were examined: all animals
- Battery of functions tested: pupillary reflex, observation of gait, extensor thrust reflex, placement reflex, righting reflex, auditory startle reflex, patellar reflex, and pain perception

IMMUNOLOGY: No
Sacrifice and pathology:
After 13 weeks of treatment, all animals were fasted overnight, weighed, anesthetized with sodium thiopental, and exsanguinated. A necropsy was performed on each animal.

GROSS PATHOLOGY: Yes
The necropsy included examination of the following: all orifices, cranial cavity, external surface of the brain, the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined whenever tissue trimming was performed, cervical tissues and organs, thoracic, abdominal, and pelvic cavities and viscera, external surface of the body, nasal cavity and paranasal sinuses
At terminal sacrifice, the following organs were weighed: adrenal, ovary, liver with drained gallbladder, lung, heart, uterus, kidney, testis with epididymis, thyroid with parathyroid, brain, thymus and spleen.

HISTOPATHOLOGY: Yes
The following tissues were histopathologically examined: adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), aorta, bone and bone marrow, brain, eye, heart, kidney, lesions, liver with gallbladder, lung, lymph node (mesenteric), mammary gland, ovary, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, testis with epididymis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus (and cervix) and vagina.
Statistics:
Mean weekly body weight, body weight change, weekly food consumption, total food consumption, clinical pathology (except hemolysis, cellular morphology gradings, and routine urinalysis data), fasted terminal body weight, and organ weight data of the treated groups were compared statistically to the data from the same sex of the control group. Tests for homogeneity of variances and ANOVA were evaluated at the 5 and 1% probability levels.
If variances of untransformed data were heterogeneous, a rank transformation of the data was performed to achieve variance homogeneity.
If the transformation did not achieve variance homogeneity, the analyses were still performed on the rank-transformed data. Control vs. treated group mean comparisons were performed at the 5 and 1% two-tailed probability levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The clincial finding of thinness in 3/8 animals treated at 1500 ppm and 5/8 animals at 2250 ppm was mainly prevalent during Weeks 7-14. The dose-related increased incidence of thinness in dogs given 1500 and 2250 ppm was attributed to the administration of the test substance.
The remaining observations are considered to be incidental findings unrelated to the treatement. Incidental findings included the following: moderate hypoactivity, vomitus discharge (yellow in color, containing food, and foamy), excessive salivation, discolored feces, liquid or mucoid feces, nonformed (soft) feces, no feces, few feces, discharge from the eye, injected sclera of the eye, alopecia, broken skin, red skin, scabs, and a scar. One female dog given 2250 ppm of the test substance had two incidents of large swollen masses located on the right shoulder in the area of the prescapular lymph node, which were accompanied with elevated body temperature and changes in haematological parameters. In addition, one male given 1500 ppm had multiple interdigital cysts and increased body temperature. For details, please refer to the attached background material.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the terminal sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Total weekly mean body weight change values (Weeks 1-13) were statistically significantly lower for males given 2250 ppm when compared to the control value. The overall weight gain of males treated at 2250 ppm showed a treatment-related depression of 72.7%, which correlated with the clinical observation of thinness. There was no effect of treatment on the weight gain of females treated at 2250 ppm and of both sexes treated at lower dose levels. The reduced overall weight gain of males treated at 1500 ppm was due to weight loss in one animal.
For details, please refer to the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower, but not statistically significant mean total food consumption values were notes for Weeks 1-5 in animals given 2250 ppm of the test substance when compared to the control. The values had improved by Weeks 7-13 and were comparable to the respective control. There was no effect of treatment on the food consumption of animals treated at 1500 ppm and lower dose levels.
For details, please refer to the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Retinal fold was noted in one female given 650 ppm of the test substance, which was present at the preinitiation examination and noted again during Week 13. However, this was not attributed to test material administration. For details, please refer to the attached background material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related alterations were observed including lower mean values for total leukocyte, neutrophil, and lymphocyte counts in animals given 2250 ppm. In males treated with 2500 ppm a statistical significant decrease of mean leukocyte and lymphocyte values were observed in Week 5. In females given 2500 ppm of the test substance, a statistical significant decrease was observed for mean leukocyte and segmented neutrophils values in Week 13.
In addition, a marked reduction in circulating platelets and panleukopenia was observed in a male treated at 1500 ppm and slight anaemia in a male at 2250 ppm, but it is unclear if these effects were treatment-related. For details, please refer to the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alterations in clinical chemistry that are attributed to the administration of the test material included statistically significantly lower mean values for alanine aminotransferase (ALT) in 1500 ppm animals (in Week 5) and in 2250 ppm females (in Week 13). Further, a statistical significant decrease was observed for mean albumin values in females at 1500 and 2500 ppm (in Week 5 and/or 13), and for mean total protein values in females given 1500 ppm (in Week 13).
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
Qualitative and quantitative urinalysis revealed no treatment-related effects at any dose level.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All animals responded within normal neurologic parameters. For details, please refer to the attached background material.
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on organ weights and ratios in either sex at any dose level. For details, please refer to the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No biologically significant macroscopic changes were noted. For details, please refer to the attached background material.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No biologically significant microscopic changes were observed. Alterations noted were consistent with commonly observed spontaneous changes in the dog strain. For details, please refer to the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponding to 19.3 mg/kg bw/day for males and 21.2 mg/kg bw/day for females
Key result
Dose descriptor:
LOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
haematology
Remarks on result:
other: corresponding to 40.9 mg/kg bw/day for males and 42.1 mg/kg bw/day for females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
other: hepatobiliary and immune system
Organ:
blood
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The study was performed under GLP conditions and similar to OECD TG 409 (adopted 1998). The study is considered reliable and valid. Under the conditions of this study, the NOAEL for subchronic toxicity was considered to be 650 ppm, corresponding to 21.2 mg/kg bw/day for males and 19.3 mg/kg bw/day for females, based on an increased incidence of thinness, slight decreases in serum albumin and total protein concentrations and reduced alanine aminotransferase activity, and additionally, reduced weight gain in males and white blood cell counts in both sexes.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Oct 1997 - 05 Apr 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
weight variation at the start of the study was > 20% of mean values, survival rates were less than 50% in 0, 150, 500 and 1500 ppm dose groups (with less than 25% in one female group at 500 ppm), continued under "Principles of methods"
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 1981
Principles of method if other than guideline:
animals were housed individually, housing temperature slightly different to the guideline
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen since it is a recommended species for chronic toxicological/cancerogenicity studies in test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., North Carolina, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 34 - 40 days
- Weight at study initiation: 105 - 194 g (males) and 113 - 162 g (females)
- Fasting period before study: none
- Housing: individually (except for the first 3 days of acclimation period) in stainless-steel cages, when health problems occurred, some animals were placed in polycarbonate cages
- Diet: certified rodent diet (#8728CM meal), Harlan Teklad, ad libitum
- Water: ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
There were no known contaminants in the diet or water at level that would have interfered with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 Oct 1997 To: 13 Oct 1999
Route of administration:
oral: feed
Details on route of administration:
DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every 2 weeks
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: at room temperature
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every 2 weeks
- Mixing appropriate amounts with (Type of food): standard diet
- Storage temperature of food: at room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of concentration:
Samples were taken from each dose preparation and stored in a freezer (-10 to -30 °C). Analyses for the concentration of the test material in the dose preparations were done by the testing facility (method MP-TK00-MA). Routine samples from all dose levels beginning with the first mix used for the study and once during month 3, 6, 9, 13, 16, 19, 22, and 25 were analyzed. All samples were stored at room temperature and analyzed within 2 weeks of preparation. The mean concentration of the dose preparation analyses for all levels ranged from 88.7 to 109% of the theoretical concentration.

Homogeneity:
Duplicate samples for homogeneity analyses were taken from the top, middle, and bottom of the dose preparations that bracket the low- and high-dose levels prepared in a prestudy mix. Mean values of the homogeneity analyses ranged from 95.9 to 96.9% and 96.0 to 100% of the theoretical concentration of 100 and 3000 ppm, respectively, thereby indicating a homogenous distribution of the test material.

Stability:
Stability was determined in a separate study at the testing facility by HPLC using a reference standard of the test item. The results of this study evaluation verified that the test article is stable in rodent diet at 25 or 5000 ppm for at least 29 days when stored at room temperature or in a freezer (-10 to -30 °C).
Duration of treatment / exposure:
at least 52 and 104 weeks
Frequency of treatment:
continously via the diet
Dose / conc.:
150 ppm
Remarks:
corresponding to 8.1 mg/kg bw/day (males) and 9.7 mg/kg bw/day (females)
Dose / conc.:
500 ppm
Remarks:
corresponding to 27.4 mg/kg bw/day (males) and 32.5 mg/kg bw/day (females)
Dose / conc.:
1 500 ppm
Remarks:
corresponding to 82.0 mg/kg bw/day (males) and 97.8 mg/kg bw/day (females)
Dose / conc.:
3 000 ppm
Remarks:
corresponding to 157 mg/kg bw/day (males) and 193 mg/kg bw/day (females)
No. of animals per sex per dose:
Terminal sacrifice (104 weeks): 60
Interim sacrifice (52 weeks): 20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on available toxicological data and the results of a
previous 13-week toxicity study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a week

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded at randomization, weekly for Week 1 through 14, and over every four weeks thereafter. Terminal body weights were recorded at the scheduled terminal sacrifice. Body weights were also recorded for animals sacrificed at unscheduled intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: Yes
Individual food consumption data were recorded weekly for Weeks 1 through 13, over four weeks the reafter and at terminal sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted
averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily during Weeks 12, 25, and 51 for animals designed for sacrafice after 52 weeks of treatment

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Each animal was examined before study initiation and before the respective scheduled sacrifices. The pupils were dilated with 0.5% Mydriacyl and the anterior portion of the eye, optic media, and ocular fundus were examined.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Weeks 13, 27, 53, 79, 103/104 (10 females/group) and 105 (10 males/group)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: collected from 10 animals per sex per group per interval (the collection taken during Weeks 13, 27 and 53 were taken from the same animals scheduled for sacrifice after 52 weeks of treatment, if possible)
- Parameters checked: haematocrit, haemoglobin concentration, erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, total and differential leukocyte count, platelet count and a measure of blood clotting time/potential, blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Weeks 13, 27, 53, 79, 103/104 (10 females/group) and 105 (10 males/group)
- Animals fasted: Yes, overnight
- How many animals: collected from 10 animals per sex per group per interval
- Parameters checked: sodium, potassium, glucose, total cholesterol, blood urea nitrogen, creatinine, total protein and albumin, globulin, albumin/globulin ratio, triglycerides, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, creatinine kinase, calcium, inorganic phosphorus, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: approximately 16 hours before blood sampling
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters checked: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood and sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional observation battery (FOB) during Weeks 66 and 67
- Dose groups that were examined: 10 animals per sex per group
- Battery of functions tested: standard cageside and open field measurements and determination of reflexes and physiological parameters
Sacrifice and pathology:
During Week 53 (20 animals per sex and group) and during Weeks 103/104 (females) and 105 (males) animals that were fasted overnight were bled for clinical pathology, anesthetized with sodium pentobarbital, weighted, exsanguinated and necropsied.

GROSS PATHOLOGY: Yes
The necropsy included a macroscopic examination of all orifices, the cranial cavity, the external surface of the brain, the external surface of the spinal cord and cut surfaces of the brain and spinal cord, the cervical tissue and organs, the thoracic, abdominal and pelvic cavities and viscera, the external surface of the body and the nasal cavity and paranasal sinuses.
The following organs were weighted: adrenal, brain, epididymis, heart, kidney, liver, lungs, ovary, pituitary, prostate, spleen, seminal vesicle, testis, thymus, thyroid with parathyroid, uterus with cervix. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues were sampled for histopathology: adrenal, aorta, brain, cecum, cervix, colon, duodenum, epididymis, esophagus, eye, femur with bone marrow, heart, ileum, jejunum, kidney, lesions, liver, lung with mainstem bronchi, lymph node (mandibular and mesenteric), mammary gland (females only), masses and associated tissues, muscle (thigh), optic nerve, ovary, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stifle joint, stomach, testis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, vagina.
Statistics:
One-way analysis of variance was used in order to analyze FOB evaluations, boy weight changes, food consumption, food efficiency, organ weights, organ-to-body weight percentages and organ-to-brain ratios.

Levene's test was done to test for variance homogeneity. In cases of heterogeneity of variance (p<=0.05), transformations were used. ANOVA was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's multiple comparison t-test was used for pairwise comparison between treated and control groups.

Group comparison were evaluated at the 5%, two-tailed probability level.

Incidence of macroscopic and microscopic observations were analyzed for group differences using Fisher-Irwin exact test.

Survival data were analyzed using log-rank test at the 5%, two-tailed probability level for group comparison. The dose-dependency of mortality was tested by the logistic regression method at the 5%, one-tailed probability level. Each tumor was classified according to the observation described by Peto et al. 1980. When the number of tumor incidences was more than eight, Peto's mortality-prevalence test was used. Otherwise, the exact permutation test was used. Rare tumors were tested at 5% upper-tailed probability level and common tumors were tested at the 1% upper-tailed probability level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Noted observations were considered to be common findings in CD rats and not related to adm
inistration of the test material. For details, please refer to the attached background material (attachment 1).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The overall adjusted survival for rats fed dietary concentration of 0, 150, 500, 1500 and 3000 ppm was 28.0, 29.3, 30.1, 40.0 and 55.0% for males, respectively and 25.3, 26.6, 20.4, 38.5 and 50.4% for females, respectively. Survival to study termination was greater in animals given 1500 and 300 ppm of the test substance compared to the control group. For details, please refer to the attached background material (attachment 2).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related alterations in mean body weights were observed over the course of the study for male rats fed 150 or 500 ppm and for female rats fed 150 ppm. 500 ppm of the test substance led to mildly reduced, but generally statistically significant body weights for females during Week 3 through 13. The lower body weights resulted from reduced food consumption and body weight gain. This effect was considered treatment-related, however, the effect was very mild and the animals recovered body weights over the course of the study.
1500 and 3000 ppm caused a reduced body weight in male and female rats. Over the course of the study, the lower body weights ranged from 4.5 to 14% and 9.3 to 21%, respectively for males and females fed with 3000 ppm when compared to the control group. The terminal body weights in the high dose group were reduced to 94 and 81% for males and females, respectively. These lower body weights were considered to be an adverse test material-related effect in rats fed with 3000 ppm of the test substance.
Lower body weight gains were also observed for animals in the top dose group. This was also considered to be test material related. The mean overall body weight gains for weeks 1 to 102 for these animals were statistically lowered to 95 and 70% of controls and considered to be an adverse finding due to the magnitude of the reductions. For details, please refer to the attached background material (attachment 3 and 14).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No apparent test material-related effects on food consumption were observed for males fed with 500 ppm and for female rats fed with 150 ppm. However, statistically significantly lower food consumption were sporadically observed for females fed with 150 ppm during Weeks 1 and 6, which were not considered to be treatment-related due to the mild and sporadic nature of the observations. Food consumption was frequently lower for males treated with 1500 and 3000 ppm and for females fed with 500 ppm. These were early effects and may represent a palatability effect. Statistically significantly lower food consumption for females treated with 1500 ppm were observed consistently from Week 1 through 21, with values ranging from 84 to 93% when compared to the control group. From week 25 through 101, the food consumption of those females
remained lower, but were not consistently statistically different from the control animals. 3000 ppm of the test substance led to statistically lower food consumption from week 1 to 53 for males and from Week 1 through 77 for females. Thereafter, the food consumption remained lower, although the values were mildly lower and only sporadically significant. For details, please refer to the attached background material (attachment 4 and 15).
Food efficiency:
no effects observed
Description (incidence and severity):
Statistical analysis was not performed, but the weekly mean food efficiency values measured over the first 13 weeks of the study, indicated that dietary administration at concentrations up to 3000 ppm had no biologically significant effect on the food efficiency. There were no apparent test materialrelated effects on food efficiency. For details, please refer to the attached background material (attachment 4).
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no obvious test material-related effects on water consumption.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related ophthalmic observations were noted at the Week 53, 103 (females) or 104 (males) examinations. Corneal keratitis and conjunctivitis were both noted at higher incidences for males and females fed 3000 ppm, but are common findings in this age and strain of rat. Therefore, theses finding are not considered to be related to the treatment. For details, please refer to the attached background material (attachment 5).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some statistically significant differences in haematological parameters were observed, however, these findings were considered incidental because they were inconsistent over time and exhibited no relationship to dose. For details, please refer to the attached background material (attachment 6 and 16).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For clinical biochemistry some statistically significant or otherwise notable differences were observed. Most of the differences were considered incidental because they were inconsistent over time and exhibited no relationship to dose. The only consistent difference was the mildly higher inorganic phosphorus for males fed with 3000 ppm which was considered due to the test material administration. Although this difference was minor, it was statistically significant at most test intervals. This effect may be related to the histopathological finding of increased incidence and severity of renal pelvis mineralization. For details, please refer to the attached background material (attachment 7 and 16).
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some statistically significant differences in urinalysis data were observed, however, these findings were considered incidental because they were inconsistent over time and exhibited no relationship to dose. For details, please refer to the attached background material (attachment 8).
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no obvious test material-related effects on FOB evaluations. Noted observations were considered to be common findings in CD rats and not related to administration of the test material.
Forelimb grip strength was statistically higher for females given 150 or 3000 ppm. This effect was not dose-dependent and not observed for males. Therefore, this effect was not considered to be treatment-related. For details, please refer to the attached background material (attachment 9).
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Interim sacrifice:
Organ-to-body weight percentage of adrenal gland, brain, heart, kidney, liver, lung, spleen and uterus were significantly greater in female rats fed with 3000 ppm when compared to the control animals. The absolute weights of these organs did not differ from the controls and no microscopic findings were observed. Therefore, these differences were considered secondary to the lower body weights of these animals. However, increased relative liver weights in females may have correlated with histipathological findings. The liver-to-body weight percentage in the females fed with 150 or 1500 ppm and the liver-to-brain weight percentage in the females given 150 ppm were also slightly greater compared to the control animals.
In males, the organ-to-body weight percentage for brain, liver and lung were significantly greater in the high dose group compared to the control group. However, the absolute organ weights were not altered compared to the control animals and no microscopic changes were observed, these effects were not considered to be toxicologically significant.

Terminal sacrifice:
The organ-to-body weight percentage of female brain, heart, kidney, liver, and lung were significantly greater in the high dose group than in the control group. However, the absolute organ weights did not differ from controls and no microscopic changes were observed. Thus, these effects were considere d to be secondary to the lower body weights in these animals. However, increased relative liver weights may have correlated with histipathological findings. The thymus-to-body weight percentage in females fed with 150 ppm was significantly lower than the controls. However, there were no differences in the absolute thymus weight compared to the control animals and no microscopic findings. Furthermore, the mean terminal body weight of these female rats were the highest, whereas the mean thymus weight of this group was the lowest. Therefore, the significantly lower thymus-to-body weight percentage was considered an incidental occurrence secondary. No significant differences in organ weights were observed between control and treated male rats.

For details, please refer to the attached background material (attachment 10 and 17).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Several macroscopic findings between the treated and the control animals. However, many of these effects were considered not to be related to the administration of the test substance because they were not observed in a dose related-manner, were considered to be related to increased survival in the treated groups, or both.
Effects that were considered related to the administration of the test substance, or had an unclear relationship to treatment included effects in the glandular stomach and kidney.

Glandular stomach:
In the high dose group, incidences of macroscopically observed dark focus/area in the glandular stomach were increased. These correlated with significantly increased incidences of hemorrhage in the glandular stomach in males fed with 3000 ppm and of erosion of the glandular stomach mucosa in females given 3000 ppm. Further, incidences of edema of the granular stomach wall were significantly increased in male and female rats fed with 3000 ppm of the test material. It was considered that these were indicative of irritant effects.

Kidney:
Granular material or calculus were observed in kidneys of a few male rats given 3000 ppm and correlated microscopically with an increased amount of pelvic mineralization in that group.

Liver:
Mottled livers were seen in males of the 1500 and 3000 ppm groups.

For details, please refer to the attached background material (attachment 11).
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Several differences were observed in microscopic findings between the treated and the control animals. However, many of these effects were considered not to be related to the administration of the test substance because they were not observed in a dose related-manner, were considered to be related to increased survival in the treated groups, or both.
Effects that had an unclear relationship to treatment included effects in the kidney (please refer to gross pathological findings), liver and in the ovary:
Instances of liver eosinophilic foci were increased in animals in the top dose group after terminal sacrifice.
Microscopically, incidences of hyperplasia of the interstitial glands of the ovary were significantly increased in female rats fed with 500, 1500 or 3000 ppm of the test substance when compared to the control animals. Interstitial gland hyperplasia was seen in ovaries undergoing atrophy with little or no active follicular development, and appeared to be an aging change. Furthermore, the incidence of interstitial gland hyperplasia for all study groups were within the range of control female rats (same strain and study duration) at the same testing facility (historical control data from 1994 to 2002). The toxicological significance of the interstitial gland hyperplasia was evaluated in an additional statement (2015, M-524671-01-1). In this statement, the ovarian lesions were considered not to be toxicologically significant due to several reasons:

1) The proliferation of stromal (interstitial) cells in the ovaries of rodents does not have a counterpart in the ovaries of human adult females and is not considered relevant for human safety assessment.
2) The incidences of interstitial gland hyperplasia were within the testing laboratory's historical control data range.
3) There is no evidence that the test substance directly affects reproductive or endocrine organs not only in the rat carcinogenicity study but in the other studies.
4) It is likely that severe suppression of body weight contributes to an increased incidence of interstitial gland hyperplasia.
5) The test substance did not affect the reproductive organs directly or have an endocrine disrupting function. It is considered likely that this finding was secondary as the result of treatment with excessiv e doses causing marked to severe suppression of body weight and associated stress, rather than any direct effect. Therefore, it is concluded that the increased incidence of interstitial gland hyperplasia observed in the treated animals is not of toxicological significance.

For details on histopathological findings, please refer to the attached background material (attachment 12 and 18).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no neoplastic lesions that were considered to be related to administration of the test material.
Three hepatocellular carcinomas were present in males fed with 500 ppm (1 at terminal sacrifice and 2 in unscheduled deaths) and in males at 3000 ppm (4 in unscheduled deaths). The incidence of hepatocellular carcinoma for males fed 3000 ppm was statistically in creased. The tumors were considered unlikely to be treatment-related owing to the absence of any dose-effect relationship and the low incidence (exceeding only slightly laboratory historical control data). Incidences and average severities of eosinophilic altered hepatocellular focus were significantly increased in rats fed with 3000 ppm of the test material. The eosinophilic foci and the hepatocellular neoplasms differed significantly in microscopic appearance and there appeared to be no relationship between the eosinophilic foci and the hepatocellular carcinomas.
In male rats, there was a significant increase of thyroid C-cell adenoma incidences in the mid- and mid-high dose group but not in the high dose group compared to the control. This was not associated with any monotonic doseresponse. Therefore, these incidences are not considered treatment-related.
In female rats there were several inconsistent increases of neoplastic lesions. They included the thyroid C-cell adenoma and carcinoma, the adrenal medulla-benign and malignant pheochromocytoma, the mammary carcinoma, adenoma and fibroadenoma. All of the above increases were not associated with significant trends and are, therefore, considered to be biologically irrelevant.
In regards to the C-cell tumors observed in female rats, an independent review of the original histopathology of the female rat thyroid was undertaken by a consensus diagnosis for each animal. This consensus pathology review confirmed that the observed thyroid C-cell adenomas were not treatmentrelated. Furthermore, it was concluded, that there was no evidence of progression of the thyroid C-cell hyperplasia to adenoma noted in the study.
Lower incidences of neoplasia were observed in males and females for e.g. thyroid follicular cell adenoma, kidney-tubular cell adenoma, pituitary adenoma and carcinomas and pancreatic islet cell adenoma and carcinoma.

For details, please refer to the attached background material (attachment 13 and 18).
Other effects:
not examined
Details on results:
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
chronic toxicity
Effect level:
150 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponding to 9.7 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
chronic toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponding to 27.4 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
chronic toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 32.5 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
chronic toxicity
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 82.0 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
The study was performed under GLP conditions and according to OECD TG 453 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus the studies are considered reliable and valid. Based on nature, incidence or chronology of neoplasms, or from the numbers of animals exhibiting tumors in treated and control rats, there was no indication of a treatment-related oncogenic effect of the test item. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 500 ppm (27.4 mg/kg bw/day) for males and 150 ppm (9.7 mg/kg bw/day) for females based on treatment related reductions in body weights and food consumption in males fed 1500 ppm and above and in females fed 500 ppm and above, and histopathology findings in males fed 3000 ppm and females fed 500 ppm and above.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Aug 1998 - 22 Mar 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
histopathology of seminal vesicle, Harderian gland and coagulating gland not performed, housing temperature with maximal 33 °C quite high
Qualifier:
according to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 1981
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: Covance Research Products, Inc., Virginia, USA
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 6 - 7 months
- Weight at study initiation: 9.3 - 12.8 kg for males and 8.5 - 11.0 kg for females
- Housing: individually housed in an elevated, stainless-steel cage measuring approximately 90 x 83 x 79 cm (d x w x h)
- Diet: PMI® Nutrition International Certified Canine Diet® Meal #5007, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
No contaminants were known to be present in the diet or water at levels, which might have interfered with this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 33
- Humidity (%): 30 - 72.1
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Sep 1998 To: 09 Sep 1999
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diets were prepared weekly
- Mixing appropriate amounts with (Type of food): basal diet
- Storage temperature of food: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples of each diet preparation were taken and stored in a freezer set to maintain -20 ± 10°C, except for one set of samples from Weeks 1 and 4, which were subjected to analytical analysis.

Concentration:
Analyses were performed on one of the duplicate samples collected in Weeks 1, 13, 26, 39, and 52. Results of routine concentration analyses conducted during Weeks 1, 13, 26, 39, and 52 indicated that all formulations were within 8% of target concentration.

Homogeneity and stability:
In a previous study, homogeneity was already demonstrated on diet formulations prepared at concentrations of 50 and 5000 ppm, respectively. These were not repeated, since the same mixing procedure and the same batch size were used. Likewise, the stability of the dietary mixture (50, 325, and 2500 ppm) kept at room temperature for up to 14 days has previously been demonstrated.
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
7 days per week
Dose / conc.:
325 ppm
Remarks:
corresponding to 7.8 mg/kg bw/day (males) and 8.5 mg/kg bw/day (females)
Dose / conc.:
650 ppm
Remarks:
corresponding to 16.6 mg/kg bw/day (males) and 15.0 mg/kg bw/day (females)
Dose / conc.:
1 500 ppm
Remarks:
corresponding to 36.3 mg/kg bw/day (males) and 40.1 mg/kg bw/day (females)
Dose / conc.:
2 000 ppm
Remarks:
corresponding to 46.4 mg/kg bw/day (males) and 52.9 mg/kg bw/day (females)
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on a 4-week study in dogs, in which a dietary concentration of 1250 ppm of the test substance was clearly a no-observable adverse effect level, but concentrations of 2500 ppm and 5000 ppm exceeded the maximum tolerated dose. In a subsequent 13-week dietary study, maximum tolerated dose (MTD) was determined to be 2250 ppm and the no-observed-adverse-effect-level (NOAEL) was 650 ppm.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: for evidence of toxic or pharmacologic effect and for evidence of mortality and moribundity (twice daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment, weekly for Weeks 1-14, once every four weeks thereafter, and at Week 53 (fasted bbody weights prior to necropsy)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: Yes
-Time schedule: Food consumption was recorded weekly for Weeks 1-13, once every four weeks thereafter, and at Week 52.

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during Week 52
- Dose groups that were examined: all animals (eyes were dilated with a mydriatic agent)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: twice prior to treatment (Weeks -2 and -1) and during Weeks 5, 13, 15 (hematology only for diagnostic purposes for one male at 2000 ppm), 26, 39, and 52
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all animals (despite Weeks 5, 13, and 15 where blood was collected only for diagnostic puprposes of one male at 200 ppm)
- Parameters checked: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood cell morphology, reticulocyte count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: twice prior to treatment (Weeks -2 and -1) and during Weeks 26, 39, and 52.
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: glucose, urea nitrogen, creatinine kinase, creatinine, total protein, albumin, globulin, albumin/globulin ratio, triglycerides, total bilirubin, total cholesterol, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, sodium, potassium and chloride

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: on the day of necropsy
- Animals fasted: Not specified
- How many animals: all surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine: twice prior to treatment (Weeks -2 and -1) and during 26, 39, and 52.
- Metabolism cages used for collection of urine: Not specified, samples were collected overnight in clean containers placed under the drainage opening of each cage.
- Animals fasted: Yes, overnight
- Parameters checked: appearance, protein, glucose, specific gravity, total volume, pH, ketones, blood, urobilinogen, bilirubin, and microscopic examination of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Weeks 26, 38, 53
- Dose groups that were examined: all animals
- Battery of functions tested: changes in skin, fur, eyes, and mucous membranes, occurrences of secretions and excretions; and changes in autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypies or bizarre behavior and changes in gait

IMMUNOLOGY: No
Sacrifice and pathology:
After at least 52 weeks of treatment, all animals were weighed, anesthetized with sodium thiopental, and exsanguinated. A necropsy was performed on each animal.

GROSS PATHOLOGY: Yes
The necropsy included examination of the following: carcass, external body orifices, abdominal, thoracic, and cranial cavities, organs/tissues
At terminal sacrifice, the following organs were weighed: adrenal, ovary, liver with drained gallbladder, lung, heart, uterus, kidney, testis with epididymis, thyroid with parathyroid, brain, pituitary, prostate and spleen

HISTOPATHOLOGY: Yes
The following tissues were histopathologically examined: adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), aorta, femur bone marrow, brain, eye, heart, kidney, lesions, liver with gallbladder, lung, lymph node (mesenteric and mandibular), mammary gland, ovary, pancreas, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, testis with epididymis, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus (and cervix), vagina.
Statistics:
Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance. Comparison tests took variance heterogeneity into consideration.
One-way analysis of variance (ANOVA) was used (if applicable) to analyze initial body weights, food consumption, continuous clinical pathology values, and organ weight data.
If the ANOVA was significant, Dunnett's t-test was used for control versus treated group
comparisons.
Group comparisons (treated groups versus control group) were evaluated at the 5.0% and 1.0% two-tailed probability level. All appropriate data collected on or after the first day of treatment, as well as the clinical pathology data obtained prior to the start of treatment, were analyzed statistically.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Redness of the skin inside the ears occurred in females treated at 1500 ppm and in males and females at 2000 ppm. The localized erythema was intermittently noted and was first observed between days 22 and 64 for the majority of the animals. Although this observation was considered to be treatment-related, it was not associated with any systemic change and did not adversely affect the animals.
The nature and distribution of other clinical observations were not indicative of any treatment-related effects. These incidental findings included: obese or thin appearance, the presence of cage sore, limited use of limbs, discharge from the ear, eyes, or urogenital region, emesis, abnormal feces (soft, mucoid, liquid, or discolored), alopecia, and skin lesions (redness, scabbing or scarring). In addition, interdigital cysts and inflammation of the external ear were observed on one or more occasions. One male at 2000 ppm was found to have a fractured tibia on day 30, which, after appropriate treatment, had healed by day 89. For details, please refer to the attached background materials (attachment 1).
Mortality:
no mortality observed
Description (incidence):
All animals survived until terminal sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of dogs given 325, 650, and 1500 ppm were unaffected by the treatment with the test substance.
A transient treatment-related weight loss occurred in males at 2000 ppm during the first week of treatment, but thereafter weight gain and terminal group mean body weight were comparable to the male controls. Females treated with the highest dose showed a slight decrease in overall weight gain during the study when compared to the control animals. Even though, these changes were attributed to treatment, they were not considered of any toxicological significance since weekly mean body weights of males and females given 2000 ppm remained comparable to those of the controls.
For details, please refer to the attached background materials (attachment 2 and 11).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For female dogs given 2000 ppm, a significantly decreased food consumption was noted during the first week of treatment. Weekly and total food consumption values for all treated males and for females treated with up to 1500 ppm were unaffected by the administration of the test substance.
For details, please refer to the attached background materials (attachment 3).
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular findings attributable to the administration of the test substance. For details, please refer to the attached background materials (attachment 4).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Slight decreases of red blood cell mass indices as hematocrit, hemoglobin and erythrocyte count were observed in the females at the top dose. In the same group, transient (Week 5, Week 39) decreases of total leukocytes, essentially caused by neutropenia were observed. On Week 5, these modifications were accompanied by a decreased monocyte and eosinocyte count, while at Week 13, only monocytemia was observed. The lymphopenia observed in the females at 650 ppm and above on week 5 was neither time- nor dose-related (pre-study sampling showed slightly lower values, while study control was relatively high).
For details, please refer to the attached background materials (attachment 5 and 11).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase of cholesterol level was observed in the females at the top dose. A decrease of alanine aminotransferase activity in both male and females at 650 ppm and above was noted. There was a clear dose-effect relationship, and a clear time-effect relationship at the high-mid and high dose.
For details, please refer to the attached background materials (attachment 6 and 11).
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the urinalysis data.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurobehavioral examinations did not reveal any changes attributable to treatment. For details, please refer to the attached background materials (attachment 7).
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The increased adrenal weight in the females at the top dose, attaining statistical significance only when expressed relative to bw, was considered treatment-related. A slight dose-related effect was apparent. Other observed modifications were not statistically significant or not dose-related.
For details, please refer to the attached background materials (attachment 8 and 11).
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic observations attributable to the treatment. For details, please refer to the attached background materials (attachment 9).
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of treatment-related microscopic changes associated with the test article administration for 52 weeks. Notable occasional findings of inflammation (lung) and of thymus lymphoid depletion (males: 1, 0, 0, 0, 1; females: 0, 1, 0, 0, 1) were not dose-related or occurred also in the control group. One animal/dose showed minimal prostate inflammation at 650 ppm and higher. Testicular atrophy and seminiferous tubular vacuolisation (moderate) was observed in 1 animal at the top dose. However, in the absence of other animals in this treatment group with similar findings, the relationship with test substance administration remains doubtful.
For details, please refer to the attached background materials (attachment 10).
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponding to 36.3 mg/kg bw/day (males) and 40.1 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 46.4 mg/kg bw/day (males) and 52.9 mg/kg bw/day (females)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
immune system
Organ:
blood
other: decreased WBC, lymphocytes, neutrophils
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The study was performed under GLP conditions and according to OECD TG 452 (adopted 1981). Notable deviations to the current OECD guideline (2018) were minor. Thus, the study is considered reliable and valid. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 1500 ppm, corresponding to 36.3 mg/kg bw/day for males and 40.1 mg/kg bw/day for females, based on transient or slightly reduced weight gain and reduced total white blood cell counts and neutrophil counts in both sexes at 2000 ppm (corresponding to 46.4 mg/kg bw/day (males) and 52.9 mg/kg bw/day (females)).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
9.7 mg/kg bw/day
Study duration:
chronic
Species:
rat
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. The study with the lowest dose descriptor was selected for endpoint conclusion.

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
Study period:
13 Apr 1999 - 13 Oct 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a common species for toxicological testing and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., North Carolina, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 240 - 317 g (males), 171 - 213 g (females)
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel cages
- Diet: Certified rodent diet (#8728C, Harlan Teklad), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days, collared for 18 h/day for 5 days before treatment

DETAILS OF FOOD AND WATER QUALITY: Diet and water was routinely checked, no signs of contaminants were found

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Apr To: 28 May 1999
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 5x5 cm
- % coverage: 10
- Type of wrap if used: gauze pad, fixed with surgical micropore tape and wrapped with Vetwrap®.
- Time intervals for shavings or clipplings: one week prior to initial treatment, one day prior to first treatment and whenever neccessary thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): sites were wiped with water-moistened paper tissues/towels
- Time after start of exposure: 6h (when exposure was terminated)

TEST MATERIAL
- Amount applied (volume or weight with unit): weight was based on the body weight of the test animal
- Constant volume or concentration used: no
- For solids, paste formed: no, but application site was moistenend with water

VEHICLE
- Justification for use: water
- Amount(s) applied (volume or weight with unit): 1 mL + water for moisturizing application site

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (flexible plastic collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material was confirmed using HPLC. It was verified that TI-435 was stable for at least 53 weeks when stored in a refrigerator set to maintain 2 to 8 °C.
Duration of treatment / exposure:
6 h/day for 29 days
Frequency of treatment:
7 days/week
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the low dose was chosen to cause no toxicity, the mid dose should cause an intermediate level of toxicity and in the high dose toxic effects should be observed but no severe skin effects or mortality meaningful evaluation
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- parameters checked: twice daily: mortality and moribundity, signs of poor health or abnormal behavior, once daily: indications of a toxic effect

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- parameters observed included changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, circling) or bizarre behavior (e.g., self-mutilation, walking backwards) and gait abnormalities.

DERMAL IRRITATION: Yes
- Time schedule for examinations: scored daily (modified Draize method)

BODY WEIGHT: Yes
- Time schedule for examinations: one day before treatment (-Day 1), on Day of first treatment (Day 1), and on Days 7, 14, 21, 28, and 30 (day of necropsy)

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 4
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count (segmented neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), blood cell morphology, reticulocyte count, activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, total bilirubin, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, magnesium, sodium, potassium, chloride, creatine kinase and sorbitol dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 prior to dosing
- Dose groups that were examined: all
- Battery of functions tested: expanded clinical observation during handling and in an open field and for sensory activity, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals were anesthetized with sodium pentobarbital after blood collection and sacrificed. Necropsy included a macroscopic examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; and organs/tissues.
Organ weights were recorded for adrenals, brain, epididymis, kidney, heart, liver, ovaries, spleen, testis, thymus and uterus.

HISTOPATHOLOGY: Yes
- Tissues were collected and preserved in 10% formalin with the following deviations:
* Fixed in Davidson's, ** preserved lobes were perfused with 10% neutral-buffered formalin, *** Fixed in Bouin's, subsequently rinsed with tap water, and transferred to 70% alcohol.
- Tissues collected: adrenal, aorta (thoracic), bone [femur (articular surface of the distal end) and sternum], bone marrow (femur and sternum), brain, cecum, colon, duodenum, epididymis, esophagus, eyes* (2, retina, optic nerve), gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lung**, mammary gland (female), lymph node (mesenteric), nasal turbinates***, ovaries, pancreas, pharynx***, pituitary, prostate, rectum, salivary gland (mandibular), sciatic nerve, seminal vesicle, skin (treated dorsal, untreated dorsal and untreated ventral inguinal), spinal cord (cervical, thoracic, and lumbar), spleen, stomach, testis***, thigh musculature thymus throidy (with parathyroid), trachea, urinary bladder and uterus
- embedding media: paraffin
- staining: hematoxylin and eosin
- animals: control and high-dose groups and from each animal that died or was sacrificed unscheduled, macroscopic lesions and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Statistics:
Homogenity of variance was checked with Levene's test. If heterogenity of of variance at p < 0.05, transformations were used to stabilize the variance. Comparison tests took variance of heterogeneity into consideration.

To analyze grip strength, motor activity, body weights, body weight changes, food consumption, food efficiency, continuous clinical pathology values, and organ weight data, an One-way analysis of variance, ANOVA, was used. If a significant difference was found, Dunnett's t-test was used for control versus treated group comparisons.

Significance was set at p ≤ 0.05
Clinical signs:
no effects observed
Description (incidence and severity):
There was no difference observed between control and treatment animals. That also included the expanded clinical observations during neurobehavioral testing.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related dermal observations noted. A single incidence of very slight edema was noted at the application site in a female given 300 mg/kg bw/day. However, since it was a sporadic finding and no dose-response was found, the observation was considered incidental.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female receiving 100 mg/kg bw/day and one female receiving 1000 mg/kg bw/day died prematurely (Day 15). In both cases, cause of death was not determinable but no treatment-related signs were found and the deaths were therefore considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Only incidental differences in body weight and body weight gains were observed. The only finding was reduced body weight changes in males treated with 1000 mg/kg bw/day in the first week (-61%) but this was not seen in any other week. However, the lower body weight gain in the firt week resulted in overall lower body weight gain in males in this treatment group throughout the study (-20% for Day 1 to 28). Sice this was due to the finding in Week 1, it was also considered incidental. In females, no differences were found for body weight and body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Sporadical differences in food consumptions were found for both sexes at the mid and high-dose treatment group. However, the differences ranged from -10.1 to +11% and no dose- or time-dependency could be established. they were therefore considered incidental.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Corresponding to the reduced body weight gain seen in week 1 for males (1000 mg/kg bw/day), the food efficiency was also reduced. As for the finding in body weight change it was considered incidental. Additionally, food efficiency in the 300 mg/kg bw/day treatment group (males) was reduced in Week 2 but this was the only interval in which this was observed and it was therefore considered incidental.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Critical effects observed:
no
Conclusions:
The effect of repeated dermal application of the test substance was analyzed in a 28 day study in rats under OECD guideline 410 and GLP. Under the conditions of the test, no toxicity was observed after repeated dermal treatment with the test substance, so that the NOAEL was set at 1000 mg/kg bw, the highest concentration tested.
The study was done according to OECD guideline 410 and under GLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
42
Species:
rat
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfill the criteria of a key study.

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
Study period:
13 Apr 1999 - 13 Oct 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a common species for toxicological testing and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., North Carolina, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 240 - 317 g (males), 171 - 213 g (females)
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel cages
- Diet: Certified rodent diet (#8728C, Harlan Teklad), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days, collared for 18 h/day for 5 days before treatment

DETAILS OF FOOD AND WATER QUALITY: Diet and water was routinely checked, no signs of contaminants were found

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Apr To: 28 May 1999
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 5x5 cm
- % coverage: 10
- Type of wrap if used: gauze pad, fixed with surgical micropore tape and wrapped with Vetwrap®.
- Time intervals for shavings or clipplings: one week prior to initial treatment, one day prior to first treatment and whenever neccessary thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): sites were wiped with water-moistened paper tissues/towels
- Time after start of exposure: 6h (when exposure was terminated)

TEST MATERIAL
- Amount applied (volume or weight with unit): weight was based on the body weight of the test animal
- Constant volume or concentration used: no
- For solids, paste formed: no, but application site was moistenend with water

VEHICLE
- Justification for use: water
- Amount(s) applied (volume or weight with unit): 1 mL + water for moisturizing application site

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (flexible plastic collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material was confirmed using HPLC. It was verified that TI-435 was stable for at least 53 weeks when stored in a refrigerator set to maintain 2 to 8 °C.
Duration of treatment / exposure:
6 h/day for 29 days
Frequency of treatment:
7 days/week
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the low dose was chosen to cause no toxicity, the mid dose should cause an intermediate level of toxicity and in the high dose toxic effects should be observed but no severe skin effects or mortality meaningful evaluation
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- parameters checked: twice daily: mortality and moribundity, signs of poor health or abnormal behavior, once daily: indications of a toxic effect

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- parameters observed included changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, circling) or bizarre behavior (e.g., self-mutilation, walking backwards) and gait abnormalities.

DERMAL IRRITATION: Yes
- Time schedule for examinations: scored daily (modified Draize method)

BODY WEIGHT: Yes
- Time schedule for examinations: one day before treatment (-Day 1), on Day of first treatment (Day 1), and on Days 7, 14, 21, 28, and 30 (day of necropsy)

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 4
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count (segmented neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), blood cell morphology, reticulocyte count, activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 5 (at termination)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, total bilirubin, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, magnesium, sodium, potassium, chloride, creatine kinase and sorbitol dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 prior to dosing
- Dose groups that were examined: all
- Battery of functions tested: expanded clinical observation during handling and in an open field and for sensory activity, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals were anesthetized with sodium pentobarbital after blood collection and sacrificed. Necropsy included a macroscopic examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; and organs/tissues.
Organ weights were recorded for adrenals, brain, epididymis, kidney, heart, liver, ovaries, spleen, testis, thymus and uterus.

HISTOPATHOLOGY: Yes
- Tissues were collected and preserved in 10% formalin with the following deviations:
* Fixed in Davidson's, ** preserved lobes were perfused with 10% neutral-buffered formalin, *** Fixed in Bouin's, subsequently rinsed with tap water, and transferred to 70% alcohol.
- Tissues collected: adrenal, aorta (thoracic), bone [femur (articular surface of the distal end) and sternum], bone marrow (femur and sternum), brain, cecum, colon, duodenum, epididymis, esophagus, eyes* (2, retina, optic nerve), gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lung**, mammary gland (female), lymph node (mesenteric), nasal turbinates***, ovaries, pancreas, pharynx***, pituitary, prostate, rectum, salivary gland (mandibular), sciatic nerve, seminal vesicle, skin (treated dorsal, untreated dorsal and untreated ventral inguinal), spinal cord (cervical, thoracic, and lumbar), spleen, stomach, testis***, thigh musculature thymus throidy (with parathyroid), trachea, urinary bladder and uterus
- embedding media: paraffin
- staining: hematoxylin and eosin
- animals: control and high-dose groups and from each animal that died or was sacrificed unscheduled, macroscopic lesions and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Statistics:
Homogenity of variance was checked with Levene's test. If heterogenity of of variance at p < 0.05, transformations were used to stabilize the variance. Comparison tests took variance of heterogeneity into consideration.

To analyze grip strength, motor activity, body weights, body weight changes, food consumption, food efficiency, continuous clinical pathology values, and organ weight data, an One-way analysis of variance, ANOVA, was used. If a significant difference was found, Dunnett's t-test was used for control versus treated group comparisons.

Significance was set at p ≤ 0.05
Clinical signs:
no effects observed
Description (incidence and severity):
There was no difference observed between control and treatment animals. That also included the expanded clinical observations during neurobehavioral testing.
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test material-related dermal observations noted. A single incidence of very slight edema was noted at the application site in a female given 300 mg/kg bw/day. However, since it was a sporadic finding and no dose-response was found, the observation was considered incidental.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female receiving 100 mg/kg bw/day and one female receiving 1000 mg/kg bw/day died prematurely (Day 15). In both cases, cause of death was not determinable but no treatment-related signs were found and the deaths were therefore considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Only incidental differences in body weight and body weight gains were observed. The only finding was reduced body weight changes in males treated with 1000 mg/kg bw/day in the first week (-61%) but this was not seen in any other week. However, the lower body weight gain in the firt week resulted in overall lower body weight gain in males in this treatment group throughout the study (-20% for Day 1 to 28). Sice this was due to the finding in Week 1, it was also considered incidental. In females, no differences were found for body weight and body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Sporadical differences in food consumptions were found for both sexes at the mid and high-dose treatment group. However, the differences ranged from -10.1 to +11% and no dose- or time-dependency could be established. they were therefore considered incidental.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Corresponding to the reduced body weight gain seen in week 1 for males (1000 mg/kg bw/day), the food efficiency was also reduced. As for the finding in body weight change it was considered incidental. Additionally, food efficiency in the 300 mg/kg bw/day treatment group (males) was reduced in Week 2 but this was the only interval in which this was observed and it was therefore considered incidental.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between control and treatment groups.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Critical effects observed:
no
Conclusions:
The effect of repeated dermal application of the test substance was analyzed in a 28 day study in rats under OECD guideline 410 and GLP. Under the conditions of the test, no toxicity was observed after repeated dermal treatment with the test substance, so that the NOAEL was set at 1000 mg/kg bw, the highest concentration tested.
The study was done according to OECD guideline 410 and under GLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
40 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfills the criteria of a key study.

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the endpoint summary.

Additional information

Repeated dose toxicity: oral administration

Data on repeated dose toxicity are available to characterize the potency of the test substance to induce toxicity after subacute, subchronic and chronic oral exposure in rodents and non-rodents.

Subacute toxicity

GLP-studies are available addressing subacute toxicity of the test substance in the species rat, mouse and dog, respectively. These studies were considered as supporting since subchronic and chronic toxicity data are also available. In order to evaluate the palatability of diets containing the test substance when administered to dogs, a palatability study was conducted.

During a 28-day toxicity study, groups of 5 rats/sex/dose were treated with 0, 1250, 2500, 5000 and 7500 ppm (M-027408-01-1) similar to OECD guideline 407 and under GLP conditions. The doses corresponded to an average test article intake of 0, 120, 249, 475 and 602 mg/kg bw/day for males, and 0, 137, 228, 454 and 686 mg/kg bw/day for females. All animals survived the treatment with the test substance up to 7500 ppm. Treatment-related clinical signs included half-closed eyes from Week 2 in all animals of the mid-high and high-dose groups and brown nasal staining from Week 3 in some animals given 7500 ppm. No clinical signs attributed to the treatment were observed among the rats given 1250 or 2500 ppm during the study. Overall, group mean body weight gain during the 4-week treatment period was significantly reduced, compared to controls, for both sexes receiving 2500, 5000 and 7500 ppm and among females receiving 1250 ppm, although this value did not attain statistical significance. The reduction in weight gain, compared to controls, followed a dosage-related trend among affected groups, with an actual overall body weight loss apparent for both sexes receiving 7500 ppm. Related to this, the overall cumulative food consumption of the groups administered 2500, 5000 and 7500 ppm showed a dose-related decrease when compared with the controls. In addition, efficiency of food utilisation revealed an impairment of food utilisation for both sexes receiving 2500, 5000 and 7500 ppm when compared to controls, reflecting the treatment-related bodyweight changes noted for these groups. Laboratory investigations on hematology, clinical biochemistry and urinalysis were performed in Week 4 of treatment. Results of the hematology investigations revealed a significant, treatment-related increase in haemoglobin concentration, red blood cell count and packed cell volume in both sexes of the groups treated at 2500, 5000 and 7500 ppm. The associated mean cell hemoglobin value was also slightly, but significantly, increased in females only. Reticulocyte counts were significantly reduced in the males treated with 5000 ppm and in both sexes treated with 7500 ppm. Furthermore, total white blood cell counts were significantly reduced in the males treated at 5000 and 7500 ppm. Treatment-related effects in clinical biochemistry included increased urea nitrogen concentrations in both sexes at 5000 and 7500 ppm, increased alkaline phosphatase (AP) activity in females at 5000 and 7500 ppm, increased glutamic-oxaloacetic transaminase (GOT) activity in both sexes at 7500 ppm, slightly increased bilirubin concentrations at 7500 ppm and increased triglyceride concentrations  in both sexes at 2500, 5000 and 7500 ppm.  With regards to urinalysis, increased urinary pH values and decreased protein values were noted for both sexes receiving 7500 ppm and males receiving 5000 ppm. Specific gravity values were decreased among all treated males (although not following a dose-related trend) and among females receiving 7500 ppm. However, in the absence of pathological changes in the kidney these functional effects are considered to be of no toxicological importance. Several statistically significant changes were noted in the absolute organ weights (pituitary, heart, lung, thyroid, ovary and brain) and in relative organ weights (heart, lung, thyroid, thymus, brain, kidney, adrenals and liver) in animals given 5000 and/or 7500 ppm. These effects are not considered to be direct effects of treatment, but are rather considered to reflect impaired weight gain and lower body weights at necropsy. Relevant macroscopic observations at necropsy were confined to the small size of some organs, notably the thymus, liver and reproductive tract of males and females treated with 5000 and 7500 ppm. Small spleen occurred in some animals treated at 7500 ppm. There were histomorphological correlates for some of the observed macroscopic lesions in the immune and reproductive systems that are considered to reflect inferred stress and markedly reduced weight gain, respectively. Generalized reduced cellularity in the splenic white pulp and thymic involutionary changes occurred at 7500 ppm. Thymic involutionary changes also occurred in some animals at 5000 ppm. Atrophy of testicular seminiferous epithelia and reduced colloid in the seminal vesicles and prostate occurred at 5000 and 7500 ppm. In females, reduced thickness of the uterine wall due to inanition occurred in some animals treated at 5000 and 7500 ppm. There were no other treatment-related histomorphological findings and no histological correlates in the liver and kidneys to account for the observed increases in plasma AP and GOT activities and increased plasma bilirubin concentration and urinary changes. In this study the NOAEL for subacute toxicity was 1250 ppm, corresponding to 120 mg/kg bw/day for males and 137 mg/kg bw/day for females, based on treatment -related reductions in body weights, reduced food consumption and hematological findings at 2500 ppm. The study was performed under GLP conditions and according to OECD TG 407 (adopted 1981). Deviations to the current version (adopted 2008) were major, as not all tissues/organs of the control animals were subjected to histopathological examination.

In a second supporting study (M-027413-01-1), groups of 6 mice/sex/dose were treated orally for at least 4 weeks with the test substance by admixture in the diet at constant nominal concentrations of 0, 500, 1000, 2000 and 4000ppm. Mean achieved dose levels were 0, 90, 190, 383 and 683 mg/kg bw/day (males) and 0, 122, 248, 491 and 619 mg/kg bw/day (females). Among mice that received 4000 ppm, 10 unscheduled mortalities occurred (4 males and 6 females) during Week 2. Four out of the six females were killed due to poor or moribund condition. All these animals showed impaired food intake and actual body weight loss in the period of treatment before death. In addition, following Week 4, two male mice given 2000 ppm died due to the stress of anaesthesia and the blood sampling procedure. These two deaths were not attributable to treatment. Clinical signs were mainly observed in both sexes given 4000 ppm and consisted of lethargy, body tremors, hunches posture, piloerection, thin/emaciated appearance, half closed eyes, unsteady gait, apparent hypothermia and pale extremities. The principal clinical signs noted among some mice receiving 2000 ppm were thin appearance and hunched posture. There were no clinical signs indicative of a reaction to treatment noted among mice of either sex receiving 500 or 1000 ppm during the 4-week treatment period. During the treatment period, actual body weight loss was recorded in both sexes given 2000 and 4000 ppm when compared with the control animals. A marginal, but not statistically significant reduction in body weight gain was also apparent in both sexes receiving 1000 ppm when compared to controls. A significantly reduced food consumption was noted in all treated groups when compared with that of the respective controls. The largest reduction in food consumption was noted in animals receiving 2000 or 4000 ppm. Consistent treatment-related haematological findings were decreased red blood cell count, hemoglobin and packed cell volume in the 2 surviving males at 4000 ppm, and reduced mean cell volume and mean cell haemoglobin values in these animals and in females at 2000 ppm. Neutrophil counts were marginally increased among all treated males. Lymphocyte counts for males receiving 4000 ppm were reduced and among females eosinophil counts were increased for mice receiving 2000 ppm compared to controls. However, total white blood cell counts were unaffected by treatment at all dose levels in both sexes and these minor changes in relative white cell numbers are considered to be stress-related and not of toxicological relevance. Male mice treated with 4000 ppm of the test article showed an increase of glutamic-pyruvic transaminase (GPT) and GOT level together with sodium, phosphorous and chloride levels whereas total protein and globin levels were decreased compared to the control animals. Among males given 2000 ppm, GOT, sodium and chloride levels were increased compared to control. In females given 2000 ppm, GPT levels were increased and triglyceride and glucose levels were decreased. Also electrolyte values for sodium and potassium were increased in female mice given 1000 and 2000 ppm, whereas chloride values were increased among all treated females when compared to the control animals. Urinalysis revealed a reduced protein level in male and female mice in a dose-related manner among all treated groups when compared to the respective controls. Marginal increase was observed for specific gravity for male mice given 4000 ppm, however, only a small volume of urine was obtained from these animals. There were no effects on organ weights that were considered a direct effect of treatment, although a number of organ weight changes at 2000 and 4000 ppm (brain, lung, thymus, testes, pituitary, spleen, adrenal, liver and ovary) were considered to reflect impaired weight gain and lower body weights at necropsy. With regards to gross pathological findings, an increased incidence of mice with small spleen and minimal adipose tissue, depressions in the stomach corpus mucosa and congestion of adrenals were observed in both sexes given 4000 ppm. In addition, the size of the thyroid, thymus and testes was reduced in males and distension of the gall bladder were observed among females, all receiving 4000 ppm. Among mice treated with 2000 ppm, an increased incidence of animals exhibiting reduced thymus size and less adipose tissue were noted in both sexes, together with reduced ovary size and a thin uterus in females. None of these changes were observed among control animals. There were histomorphological correlates for some of the observed macroscopic lesions. Some animals given 2000 or 4000 ppm showed changes in the immune system (reduction of cellularity of the white pulp and atrophy of red pulp in the spleen), together with thymic involution. Furthermore, changes in the male and female reproductive tract (atrophy of testicular seminiferous epithelia, reduced colloid within seminal vesicles and prostate, uterine wall thickness and reduced presence of corpora lutea) were noted among animals from the high and high-intermediate dose levels. All these changes were considered to reflect inferred stress and markedly reduced weight gain. There were no treatment-related histomorphological correlates in the liver and kidneys to account for the observed increases in plasma transaminase activities and electrolyte concentrations. Similarly, there were no histomorphological correlates for the gross lesions noted at necropsy in the gall bladder, adrenals and stomach. The NOAEL for subacute toxicity was considered to be 1000 ppm, corresponding to 190 mg/kg bw/day for male and 248 mg/kg bw/day for female mice. This was based on clinical signs of toxicity, weight loss, hematological and blood biochemistry perturbations and histomorphological lesions in the spleen and ovaries observed at 2000 ppm. The study was performed under GLP conditions and according to OECD TG 407 (adopted 1981). However, deviations to the current version (adopted 2008) were major, as not all tissues/organs of the control animals were subjected to histopathological examination.

For evaluating the palatability of diets containing the test substance at concentrations up to 5000 ppm when administered to dogs, a study on beagle dogs was conducted (M-027385-01-1). Female dogs were assigned to 2 groups. Animals in Group 1 were given the basal diet only for Days 1 through 11, whereas dogs in Group 2 were given diets containing the test substance at a concentration of 3000 ppm (Days 1 through 3), 4000 ppm (Days 4 through 8) and 5000 ppm (Days 9 through 11). Mean achieved dose levels for the treated group were 51.1, 50.8 and 51.8 mg/kg bw/day for days 1 – 3, 4 – 8 and 9 – 11, respectively. There were no deaths during the study and no treatment-related clinical signs were observed. The animals given 3000 ppm and then 4000 ppm during days 1 – 8 showed a progressive decrease in mean body weight of 0.85 kg compared to a mean weight increase in the controls of 0.25 kg. The mean weight of the treated animals decreased further by 0.25 kg from day 9 – 11, compared with a mean control weight loss of 0.1 kg. In addition, the mean food consumption of treated animals decreased as the test substance concentration in the diet increased. At the 3000 ppm diet concentration, mean daily food consumption was 65% (35% lower) of that of the control. At the 4000 ppm diet concentration, mean daily food consumption was 43% (57% lower) of that of the control animals. At the 5000 ppm diet concentration, mean daily food consumption was 37% (63% lower) of that of the control group. The no-observed-effect-level (NOEL) was considered to be 5000 ppm, which was equivalent to a dose level of 51.8 mg/kg bw/day, based on the absence of treatment-related clinical signs. Since a concentration-related decrease in food consumption was observed, diets containing the test substance at concentrations ≥ 3000 ppm are less palatable to the dog than untreated diet. Therefore, weight loss in treated animals is considered not to be a direct expression of toxicity, but to reflect reduced food consumption as a consequence of reduced diet palatability. The maximum dietary concentration of 5000 ppm should be selected to confirm that improved acceptance of this dietary concentration may occur, thus allowing evaluation of the potential toxicity the test item at dietary concentrations up to 5000 ppm.

In a third supporting study (M-027342-01-1), four groups of 3  beagle dogs/sex/dose were treated orally with the test substance by admixture in the diet at constant nominal dietary concentrations of 0, 1250, 2500 and 5000 ppm for at least 4 weeks. After 3 weeks of treatment, the surviving animals at 5000 ppm were killed due to deteriorating health. Mean achieved dose levels were 0, 36.3, 35.8 and 62.4 mg/kg bw/day (males) and 0, 35.6, 52.3 and 57.4 mg/kg bw/day (females). No unscheduled death occurred among the animals of the control and low-dose group. Among the animals given 2500 ppm, one male was found dead (Day 18) and one female was sacrificed in extremis (Day 32). Among the animals given 5000 ppm, one male was found dead (Day 19) and one male and one female were sacrificed in extremis. Due to declining health, the remaining three animals in the high-dose group were killed on Day 22 (designated first interim sacrifice). Treatment-related clinical signs began on Day 18 in some animals given 2500 or 5000 ppm and progressed quickly. These signs were mainly limited to the animals that died or were killed in extremis and included various combinations of prostration or hunched posture, discoloured and mucoid faeces, salivation, dyspnea or polypnea, sanguineous nasal discharge, thin appearance, pale gums and dehydration. There were no treatment-related clinical signs at 1250 ppm. Males given 2500 and 5000 ppm and females treated with 5000 ppm showed markedly reduced body weight gain throughout the study. This was statistically significant from Week 1 - 3. Body weight changes for Week 1 - 4 and 1 - 5 were not statistically analyzed for males given 2500 ppm and for all animals treated with 5000 ppm, due to the small sample size (N < 2).The slightly reduced weight gain of animals treated with 1250 ppm was not statistically significant and occurred in the absence of other treatment-related effects. Therefore, for the low-dose group it is considered to be a non-adverse effect. Food consumption was markedly reduced throughout the study for both sexes treated at 5000 ppm and for males at 2500 ppm. Females treated with 2500 ppm showed reduced food consumption during the first 2 weeks of treatment only. The food consumption of both sexes at 1250 ppm was slightly reduced but was considered not to be an adverse effect of treatment. No ophthalmoscopic abnormalities were detected during the course of the study that could be attributed to test material administration. Treatment-related changes in the clinical hematology data occurred in animals given 2500 and 5000 ppm: decreases in platelet, total leukocyte, segmented neutrophil, and lymphocyte counts and erythrocyte mass (erythrocyte count, hemoglobin, and hematocrit). No treatment-related hematology findings were observed in animals treated with 1250 ppm.  Reduced plasma concentrations of glucose (5000 ppm males only), alanine aminotransferase activity, total protein, albumin, globulin, inorganic phosphorus and chloride ion were observed at 2500 and 5000 ppm, and increased plasma alkaline phosphatase activity, total cholesterol and triglyceride concentrations were also primarily observed at 5000 ppm. No treatment-related clinical chemistry findings were observed in animals treated with 1250 ppm. The urinalysis results were generally unremarkable and comparable between the groups, with the exception that a few incidences of occult blood and erythrocytes was noted in animals given 1250 and 2500 ppm in Week 5. The source of the hematuria is not apparent from the data examined, but is unlikely to be treatment-related. Generally, absolute and relative organ weights (brain, kidney, testis, liver and thyroid) for animals of the high-dose group were lower, reflecting the treatment-related depression of body weight gain. There were no treatment-related macroscopic findings in the survivors at 2500 and 5000 ppm or in any animal treated at 1250 ppm. Four of the 5 animals treated at 2500 and 5000 ppm that died showed mottled lungs at necropsy with histological evidence of congestion, hemorrhage or oedema in 3 animals. However, these findings are considered to be a consequence of opportunistic infection that developed in the presence of declining health of the animals and treatment-related depression of the lymphoreticular/hematopoietic system. None of the other macroscopic findings (enlarged liver, dark areas in stomach and urinary bladder) in these animals had histological correlates. Treatment-related histopathological findings were observed in animals given 2500 or 5000 ppm, but not in animals treated with 1250 ppm. Corresponding to the decreased mean hematology values, a treatment-related effect on hematopoietic/lymphoid tissues was histologically evident in in the mid- and high-dose group, with sternal and femoral bone marrow exhibiting a diffuse hypocellularity as a result of variable degrees of loss of hematopoietic elements with relatively increased amounts of fat. In some animals of the high-dose group as well as in the animals of unscheduled deaths given 2500 ppm, congestion of marrow sinusoid with evidence of hemorrhage in the sternal and femoral bone marrow was observed. In addition, there was noticeable depletion of lymphoid cells within the thymus, spleen, and mesenteric lymph nodes of animals given 2500 or 5000 ppm. A further treatment-related finding, which may indicate a direct effect from the test substance, was a subtle increase in the incidence and degree of crypt gland dilatation within the duodenum of animals given 2500 or 5000 ppm, which was often accompanied by a few necrotic epithelial cells within the affected gland. Histopathological findings in the lung for three of the unscheduled deaths were most probably indirect effects of poor health. The NOAEL for subacute toxicity was 1250 ppm, corresponding to 34.3 mg/kg bw/day for male and 35.8 mg/kg bw/day for female beagle dogs. This was based on the occurrence of clinical signs of toxicity, weight loss, hematological and blood biochemistry changes and histomorphological lesions in hematopoietic/lymphoid tissues and duodenum at 2500 ppm. The study was performed under GLP conditions and according to OECD TG 409 (adopted 1984). Notable deviations from the OECD guideline included the short study duration of 28 days and the low animal number of 3 animals per sex and dose. However, the study is considered reliable and valid.

Subchronic toxicity

Key studies are available addressing the subchronic toxicity of the test substance in the species rat and dog, respectively.

In the first key study (M-027268-01-1) four groups of 15 rats/sex/dose were treated orally for at least 13 weeks with the test substance by admixture in the diet at constant nominal concentrations of 0, 150, 500, and 3000 ppm. Two additional recovery groups of 15 rats/sex, similarly treated for 13 weeks at 0 and 3000 ppm, were maintained on untreated diet for 7 weeks prior to necropsy. The stability, homogeneity and achieved concentrations of test diets were satisfactory. Mean achieved dose levels were 0, 9.0, 27.9, and 202 mg/kg bw/day (males) and 0, 10.9, 34.0 and 254 mg/kg bw/day (females). Survival of the treated animals was comparable to control animals. One male rat given 500 ppm was found dead on nominal Day 17. The Cause of death was undetermined. Clinical and/or cageside observations attributable to exposure to the test substance were not observed. The Body weight gain remained unaffected in both sexes at doses up to and including 500 ppm. Animals given 3000 ppm showed a reduced body weight gain for both sexes of approximately 11 and 12% in the male and female, respectively, compared with the control. The food consumption and utilization was unaffected in both sexes at all doses tested. Pre- and post-exposure ophthalmological examinations provided no evidence that the test substance induced ophthalmic toxicity in either sex at any dose tested. There were no treatment-related effects on the hematological and urinalysis profiles in either sex at any dose level. The results of clinical biochemistry parameters showed increased hepatic enzyme activities at the highest dose tested. These included cytochrome P-450, aminopyrine N-demethylase, and p-nitroanisole O-demethylase in both sexes, and ethoxyresorufin O-deethylase, and pentoxyresorufin O-dealkylase in males only. However, these changes may simply reflect an adaptive response by the liver to an increased need to facilitate the metabolism and excretion of an exogenously administered test substance. Morphological evidence of a direct toxicological effect of the test item on the liver was not observed. Relative organ weight increases were seen for brain, kidney, and testis of 3000 ppm males and for brain and heart of 3000 ppm females at the 13-week termination. Relative organ weight increases were also present in 3000 ppm recovery females for brain, lung, spleen, and ovaries. These variations were considered secondary due to alterations in body weight gain observed in both sexes at the highest dose tested. This conclusion was also supported by the lack of microscopic evidence of direct toxicological effect of the test item on any tissue examined in this study. Even though, there were no treatment-related gross lesions at necropsy, treatment-related histomorphological alteration occurred in the spleen of males treated at 3000 ppm. A statistically significant increased incidence of pigmentation was observed in the spleen of 3000 ppm males (13/15) compared to the control animals (6/15). This was suggested to be deposition of hemosiderin, an iron-containing pigment derived from the hemoglobin of disintegrating red blood cells. By conclusion of the recovery period, reversibility was suggested in every parameter that had been influenced and with exception of the body weight gain, parameters had fully reverted to control levels. In summary, under the conditions of this study, the NOAEL for subchronic toxicity was 500 ppm, corresponding to 27.9 mg/kg bw/day for male and 34.0 mg/kg bw/day for female rats. This was based on the reduced body weight gain in both sexes, and hepatic microsomal enzyme induction in both sexes at 3000 ppm and pigmentation of the spleen in males only at 3000 ppm. The study was performed under GLP conditions and according to OECD TG 408 (adopted 1981). Deviations from the current OECD guideline (2018) were related to detailed clinical observations, specific blood parameters and specific organ weights. However, the study is considered reliable and valid.

In the second key study (M-036499-02-1) five groups of 4 beagle dogs/sex/dose were treated orally with the test substance by admixture in the diet at constant nominal dietary concentrations of 0, 325, 650, 1500 and 2250 ppm for at least 13 weeks. Mean achieved dose levels were 0, 9.2, 19.3, 40.9 and 58.2 mg/kg bw/day (males) and 0, 9.6, 21.2, 42.1 and 61.8 mg/kg bw/day (females). Chemical analysis of diets indicated adequate stability, homogeneity and achieved concentrations. All animals survived to the terminal sacrifice. The dose-related increased incidence of thinness in dogs given 1500 and 2250 ppm was attributed to the administration of the test substance. This was mainly prevalent during Weeks 7-14. The remaining observations were considered to be incidental findings. One female dog given 2250 ppm of the test substance had two incidents of large swollen masses located on the right shoulder in the area of the prescapular lymph node, which were accompanied with elevated body temperature and changes in hematological parameters. Due to antibiotic therapy, the animal was returning to normal by Day 68. One male given 1500 ppm had multiple interdigital cysts and increased body temperature. Even after treatment of the cyst with local cleaning of the sites, the animal appeared unthrifty (unhealthy) through the final weeks of the study. Total weekly mean body weight change values (Weeks 1-13) were statistically significantly lower for males given 2250 ppm when compared to the control value. The overall weight gain of males treated at 2250 ppm showed a treatment-related depression of 72.7%, which correlated with the clinical observation of thinness. There was no effect of treatment on the weight gain of females treated at 2250 ppm and of both sexes treated at lower dose levels. The reduced overall weight gain of males treated at 1500 ppm was due to weight loss in one animal. Lower, but not statistically significant mean total food consumption values were notes for Weeks 1-5 in animals given 2250 ppm of the test substance when compared to the control. The values in Weeks 7-13 were comparable to the respective control. There was no effect of treatment on the food consumption of animals treated at 1500 ppm and lower dose levels. The neurological and ophthalmological examinations did not reveal any treatment-related effects in either sex at any dose level. Treatment-related alterations were observed in hematological parameters including lower mean values for total leukocyte, neutrophil, and lymphocyte counts in animals given 2250 ppm. In addition, a marked reduction in circulating platelets and panleukopenia was observed in a male treated at 1500 ppm and slight anaemia in a male at 2250 ppm, but it is unclear if these effects were treatment-related. Clinical biochemistry findings observed at one or more treatment intervals included lower alanine aminotransferase and lower albumin levels in animals given 1500 and 2250 ppm as well as lower total protein values in females given 1500 ppm and in all animals given 2250 ppm. Qualitative and quantitative urinalysis revealed no treatment-related effects at any dose level. There were no treatment-related effects on organ weights and ratios or on the nature and incidence of macroscopic lesions at necropsy in either sex at any dose level. No biologically significant macroscopic changes were noted. In addition, also no biologically significant microscopic changes were observed. Alterations noted were consistent with commonly observed spontaneous changes in the dog strain. In total, under the conditions of this study, the NOAEL for subchronic toxicity was 650 ppm, corresponding to 21.2 mg/kg bw/day for male and 19.8 mg/kg bw/day for female dogs. This was based on an increased incidence of thinness; slight decreases in serum albumin and total protein concentrations and reduced alanine aminotransferase activity; reduced body weight gain in males; and white blood cell counts in both sexes. The study was performed under GLP conditions and according to OECD TG 409 (adopted 1981). Thus, the study is considered reliable and valid.

In addition to the two key studies in the species rat and dog, two subchronic studies are available assessing the oral toxicity of the test substance in rats and mice. However, serious GLP problems were found at the testing facility during the conduct of these 13-week studies. Consequently, the studies are both considered unreliable and not adequate for the toxicological evaluation of the test substance, and are therefore flagged as disregarded studies. For the sake of data completeness, both studies are shortly discussed below.

In the first disregarded study ten rats/sex/dose were administered the test substance at daily dietary levels of 0, 100, 250, 1250 and 2500 ppm, corresponding to a mean achieved compound intake of 0, 7.7, 19.7, 96 and 189 mg/kg bw/day in males and 0, 9.7, 24.0, 119, 232 mg/kg bw/day in females, respectively (M-031643-02-1). There was no mortality and no treatment-related findings in clinical signs and during the ophthalmoscopic examination. The main treatment-related changes consisted of marginal impairment of food consumption and a slight impairment of body weight gain during the first two weeks of treatment in both sexes receiving 2500 ppm and females receiving 1250 ppm. The main findings with regards to clinical chemistry were slightly increased red blood cell parameter values among males receiving 2500 ppm; together with slightly increased female alkaline phosphatase values and male triglyceride values among rats at this dose level. Slight changes in the urinalysis parameters were noted among animals receiving 1250 and 2500 ppm. Changes in relative organ weights were increased liver weights for both sexes treated with 2500 ppm, which may account for the increased alkaline phosphatase and triglyceride values noted in this dose group. Also among females, slightly increased relative spleen, uterus and ovary weights were noted for animals receiving 1250 and 2500 ppm and increased adrenal weights for animals receiving 2500 ppm. The increase in uterine weight was partly corroborated by both gross pathological and histopathological findings (uterus fluid distention/ uterus luminal dilatation). These results suggested possible endocrine mediated effects on reproductive organs in females. Therefore, the findings in the uterus, ovaries and adrenals were re-evaluated focused on fluid distention of uterus during necropsy / histopathology, on the reproductive cycle stage of the animals and its relationship to uterine dilations and on the increase in corpora lutea / histopathology of the ovary (position paper M-031166-01-1). The re-evaluation revealed that there was an increased number of animals in proestrus at all treated dose levels, which was in line with the uterine dilation. Further, an increase in number of corpora lutea at 2500 ppm was noted, which did not reach statistical significance. The statistically significant increase in relative adrenal weights were not accompanied by morphological changes attributed to the test compound. The evaluation of the histopathology revealed that the adrenal tissues were damaged during necropsy and that a complete evaluation of the compound effects on adrenals cannot be ascertained. These effects could be associated with the stress to the animals due to handling and co-housing. Based on the evaluation of uterus and ovarian findings a possible compound effect on hormonal regulation was stated even though the testing facility concluded that uterus and ovarian weights were within their historical control range. However, the present study is considered to be unreliable owing to serious GLP issues and according to the position document (M-031166-01-1) these effects were not reproducible. More recent repeated-dose toxicity studies on rats showed no evidence of the test item having endocrine properties.  In the subchronic rat study special emphasis was given to assess the potential effects on the uterine weights by selecting animals for terminal sacrifice which were in the same estrous cycle stage to the extent possible. In this study, no effect on uterus, ovaries, or adrenals were noted. In addition to the subchronic study the results of the 2-generation reproduction toxicity study (performed according to OECD guideline 416 and under GLP conditions) showed no effects on mating, fertility, gestation, birth, live birth, viability, or lactation indices in either generation. No effects were observed on the estrous cycle (number or length) or ovarian follicular development. In males sperm count, sperm morphology and fertility were not affected. No compound-related histological changes were noted in reproductive organs or tissues in the study. Results of both the subchronic studies in the rat and mouse, and the 2-generation reproduction toxicity study in the rat clearly demonstrated that the test substance does not show endocrine properties. These conclusions are further supported by the fact that in the rat chronic/oncogenicity study no evidence of compound-related oncogenicity (including the endocrine organs) was observed up to, and including, the highest doses tested, which was or exceeded the maximum tolerated dose.

In the second disregarded study ten mice/sex/dose were treated with the test substance at daily dietary levels of 0, 100, 500, 1000 and 1500 ppm, corresponding to a mean achieved compound intake of 0, 16, 82, 160 and 263 mg/kg bw/day in males and 0, 22, 107, 207, 329 mg/kg bw/day in females, respectively (M-031652-02-1). There were no treatment-related findings with regards to mortality, ophthalmoscopic examination, hematology, urinalysis and macroscopic pathology. However, 12 mice were found dead following the blood sampling procedure in Weeks 12 and 13 of treatment. Clinical signs which were considered to be related to the treatment included vocalization noted from Week 4 of treatment (4/10 males and 6/10 females given 1500 ppm). Significantly reduced group mean body weight gain was noted among both sexes given 1500 ppm. In addition, marginally reduced group mean body weight gain was observed for males receiving 100, 500 and 1000 ppm and for females receiving 1000 ppm. Subsequently body weight gain for both sexes given 1000 and 1500 ppm improved slightly and was slightly lower than in the control group during Week 2 to 13 of treatment. Body weight gain of all other groups was comparable to the controls during Weeks 2 to 13.  The food consumption of female mice receiving 500, 1000 and 1500 ppm was significantly reduced during the first two weeks of treatment in comparison with the control animals. During Weeks 3 to 13 of treatment food consumption was comparable with that of controls for all treated groups. Food utilization was impaired for both sexes given 1500 ppm and to a lesser extent for animals receiving 1000 ppm when compared to the controls during the treatment period. Clinical biochemistry changes included an increase in alkaline phosphatase values for female mice given 1000 and 1500 ppm together with glutamic oxaloacetic transaminase values among all treated female groups. However, no dosage-related trend was apparent for these parameters. Further, sodium values were slightly increased for females given 1000 and 1500 ppm, together with phosphorous values for females given 500, 1000 and 1500 ppm and potassium values for female mice receiving 1500 ppm. Decreased urea nitrogen values were noted for both sexes given 1500 ppm, together with slightly decreased total protein values for females given 500, 1000 and 1500 ppm and globulin values for female mice given 1500 ppm. Relative organ weights showed a significant reduction in group mean kidney weights among all male treated groups. However, this was not following a dose-related trend. A reduction in group mean lung weights among females which received 1500 ppm was noted. Microscopic examination revealed an increased incidence of female mice showing sparse number of corpora lutea and an increased prominence of uterine endometrial glands in animals given 1500 ppm. These results suggested possible endocrine mediated effects on reproductive organs in females. Therefore, the findings in the uterus and ovaries were re-evaluated (position paper M-031166-01-1). The re-evaluation revealed that there was an increased incidence of sparse number of corpora lutea and a significant decrease of the number of corpora lutea and follicles on both ovaries among females given 1500 ppm when compared to the control animals. Further, a significant increased number of animals in estrus was noted among female mice given 1500 ppm in comparison to the control animals. In line with the 90-day rat study (M-031643-02-1) the uterine and ovarian findings showed a possible compound effect on the hormonal regulation. However, the present study is considered to be unreliable owing to serious GLP issues and in the position document (M-031166-01-1) it was concluded that these effects were not reproducible and not supported by results of a further repeated dose toxicity studies in the mouse. These conclusions are further supported by the fact that in the mouse carcinogenicity studies no evidence of compound-related oncogenicity (including the endocrine organs) was observed up to, and including, the highest doses tested, which either met or exceeded the maximum tolerated dose) in mice. As serious GLP problems were found at the testing facility during the conduct of both 13-week studies with rats and mice both the studies were discontinued. The results of the studies on endocrine related effects were not supported by results of GLP-compliant repeated dose toxicity studies in rats, mice and dogs. The more recent, GLP-compliant, studies showed no evidence of the test substance having endocrine properties.

Longterm toxicity was evaluated in beagle dogs according to OECD 452 and GLP. Briefly, 4 dogs per dose and sex were exposed over 52 weeks to the test item vie the food at dose levels of 325, 650, 1500 and 2000 ppm, corresponding to 7.8, 16.6, 36.3 and 46.4 mg/kg bw/day in males and 8.5, 15.0, 40.1 and 52.9 mg/kg bw/day in females. Dose levels were selected based on the subacute (M-027342-01-1) and subchronic toxicity study (M-036499-02-1). Homogeneity and stability of the test item in the food were demonstrated in previous studies. Animals were observed at least twice daily for mortality and moribundity, and daily for abnormalities and signs of toxicity. In addition, a thorough physical examination was conducted weekly. Body weights were measured weekly for Weeks 1-13, every 4 weeks thereafter, and at Week 53. Food consumption data were collected weekly for Weeks 1-14, every 4 weeks thereafter, and at Week 52. Neurological examinations were conducted during Weeks 26, 38 and 53. Ophthalmic examinations were conducted before initiation of treatment and during Week 52. Blood and urine samples were collected for clinical hematology, clinical chemistry and urinalysis tests twice prior to initiation of treatment and during Weeks 5 (hematology only), 13, 15 (one diagnostic sample only), 26, 39, and 52. After 52 weeks of treatment, animals were weighed, anesthetised, exsanguinated and necropsied. At necropsy, macroscopic observations were recorded, selected organs were weighed and selected tissues were collected and preserved. Microscopic examinations were conducted on tissues from each animal. The dietary administration of TI-435, at dosages of 325, 650, 1500 and 2000 ppm for up to 52 weeks did not produce treatment-related and/or adverse effects on neurobehavioral evaluations, ocular examinations, coagulation parameters, urinalysis or organ weight data. Doses of 2000 ppm were associated with a transient weight loss in males during the first week of treatment, as well as with a slightly reduced growth rate (Week 1-53) and a marginally lower food intake for Week 1 in females. Localized erythema inside the ears was noted more frequently in dogs given 2000 ppm but this local change and small body weight differences did not adversely affect the animals. Transient decreases in total leukocyte, and neutrophils and alanine aminotransferase values were also observed at 2000 ppm, with no apparent mechanism. Although the decrease in total leukocytes was less severe than observed in previous studies, at least two dogs given 2000 ppm had leukocyte counts below the normal range. Changes in lymphocyte counts were not attributed to treatment since lower counts were observed during the pre-treatment period. These findings were not associated with any macroscopic and microscopic changes, but the leukopenia observed at 2000 ppm suggests that the immune system is the likely target organ of toxicity in this study. Treatment-related alterations associated with dosages of 650 and 1500 ppm were limited to decreases in alanine aminotransferase (ALT) values. Although decreases in alanine aminotransferase were consistent with that seen in previous studies, this finding was not supported by concurrent changes in any other parameters (e.g. decreased food consumption, serum chemistry changes and microscopic lesions). Dogs were unaffected by decreases in alanine aminotransferase. Localised erythema was frequently seen in females given 1500 ppm but this finding was not associated with any systemic change and did not adversely affect the animals. Based on the obtained results, 1500 ppm was considered as NOAEL (36.3 mg/kg bw/day in males and 40.1 mg/kg bw/day in females) based on anaemia, decreased WBC parameters and decreased serum ALT at the next higher dose level of 2000 ppm (46.4 mg/kg bw/day in males and 52.9 mg/kg bw/day in females).

Chronic toxicity

Data on chronic toxicity are available based on carcinogenicity studies performed in the rat and the mouse.

In summary, a combined chronic toxicity/carcinogenicity study was performed under GLP conditions and according to OECD TG 453 (adopted 1981) in Wistar (Crl:CD(SD)IGS BR) rats. Under the conditions of this study, the NOAEL for chronic toxicity was considered to be 500 ppm (27.4 mg/kg bw/day) for males and 150 ppm (9.7 mg/kg bw/day) for females based on treatment-related reductions in body weights and food consumption in males fed 1500 ppm and above and in females fed 500 ppm and above, and histopathology findings in males fed 3000 ppm and females fed 500 ppm and above.

In mice, carcinogenicity was tested according to OECD TG 451 (adopted 1981) and GLP. Based on treatment-related reductions in body weights, body weight gain and food consumption, a chronic NOAEL of 350 ppm was derived for male and female mice which corresponds to 47.2 mg/kg bw/day for males and 65.1 mg/kg bw/day for females.

For further details, please refer to the information provided in section “Carcinogenicity” (chapter 7.7 in the technical dossier and chapter 5.8 in the CSR).

Repeated dose toxicity: dermal administration

In a 4-week repeated dose study, the dermal toxicity potential of the test substance to the rat was assessed (M-025976-01-1). Ten rats/sex/dose received 0, 100, 300 and 1000 mg/kg bw test substance per day for 4 weeks. The test substance was moistened with water and applied to shaved   skin for 6 - 6.5 h/day under occlusive conditions. During this time, animals wore a plastic collar to prevent ingestion of the test item. The treatment was repeated 7 times per week for 4 weeks. The control group (10 male/10 females) received the vehicle (water) only. Animals were checked daily for clinical signs, food consumption was calculated as mean intake per day and the body weight was recorded weekly. In week 4, blood was drawn for haematology and clinical chemistry investigation. An opthalmoscopic examination was performed and the rats were subjected to a neurobehavioural examination in week 4.  Gross necropsy was performed and histopathological examination was performed on selected tissues.

Two females (100 and 1000 mg/kg bw/day) died on Day 15. In both cases, the cause of death was not determinable but no treatment-related signs were found and the deaths were therefore considered incidental. No local or systemic treatment-related toxicity was observed for any parameters. The NOAEL systemic and local for the test material was set to 1000 mg/kg bw/day. The study was conducted according to OECD guideline 410 and under GLP conditions.

Conclusion:

In summary, the subacute, subchronic and chronic toxicity studies in rodents and dogs indicated that the hematopoietic and lymphoid organs as possible targets for repeated dose toxicity of the test substance. This was particularly evident at doses associated with systemic toxicity characterized by decreased body weight. The lowest relevant NOAEL for repeated oral exposure was 27.9 mg/kg bw/day in the 90 day, and 9.7 mg/kg bw/day in the 2-year study in rats. In the dog, the lowest relevant NOAEL was 19.3 mg/kg bw/day in the 90-day study, and 36.3 mg/kg bw/day in the 52-week toxicity study. The test substance exhibited very low toxicity by the dermal route in a 4-week study in the rat. The systemic dermal NOAEL was 300 mg/kg/day on the basis of the slight body weight effects in males at 1000 mg/kg bw/day. However, no local effects on the skin were observed.

References not included in IUC:

M-027385-01-1, Moore, M.R. 1998: Pilot palatability study for dietary concentrations of TI-435 in dogs, Testing laboratory: Covance Laboratories Inc., Virginia, USA, Report no.: 6155-107, Owner company; Bayer AG, Study number: DTOX015, Report date: May 01, 1998

Justification for classification or non-classification

An extensive data base exists to assess repeated dose toxicity following the oral and dermal route. Based on the available data on short- and longterm exposure, the test substance does not meet the classification criteria in regard to STOT-RE for neither route according to the CLP regulation (EC) No. 1272/2008 which is in line with the harmonized classification of the test item according to Annex VI of the CLP Regulation (EC) No. 1272/2008.