Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July, 2010 - 06 April, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch No.: E09000602
Appearance: white solid
Storage: tightly closed at room temperature

Test animals

Species:
rat
Strain:
other: Rat, Crl:WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
Age at delivery 8 weeks
Source: Charles River, Germany

Body weight range at acclimatization
Males: 236 ( 217 – 258 )g
Females: 169 ( 154 – 180 )g

Acclimatization
For 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HOUSING & DIET
--------------
The animals were housed according to the German Animal Welfare Guidelines for housing
of Laboratory Animals (e.g. Official Journal of the European Union L 197/1 Commission
recommendation of 18 June 2007 on guidelines for the accommodation and care of animals
used for experimental and other scientific purpose).
The animals were gang-housed (2 or 3 animals/sex) in type IV Makrolon® cages. They
were provided with means to hide. During the short time period between functional
observations and motor activity, the animals were kept individually in type III Makrolon®
cages.
Separate animal rooms are used for each study to avoid cross-contamination between
different studies. The animal rooms and experimental animal studies are regularly
inspected by the animal welfare authorities. The conditions have been approved by these
authorities.
Softwood granulate analyzed periodically for contaminants served as the cage litter served
as the cage litter (supplier: ABEDD LAB&VET Service GmbH, 1160 Wien, Austria). The
cages were color labeled indicating the different dose groups. The same colors were used
for labeling the treatment equipment and the formulation containers.
Lighting: by fluorescent tubes: 12 hour light/dark cycle provided via automatic timer.
The rats were fed Provimi Kliba 3433.0 diet. They received tap water ad libitum. Drinking
water was available from Makrolon® drinking bottles freshly filled twice a week.
Analyses of food and water for contaminants: The diet was checked periodically by an
independent and approved laboratory, according to the specifications of the manufacturer.
Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins,
pesticides, and antibiotics (latest food analysis in appendix 3). The drinking water
employed in this type of study is regularly analyzed microbiologically, physicochemically,
and chemically

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Details on oral exposure:
The test item formulations in 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) were prepared daily. The preparations were stored under normal room conditions. Exposure to light was kept to a minimum.

The test material was administered orally by gavage. The control rats received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), at the same frequency as the animals treated with the test item.

Administration was done orally by gavage, once daily, 7 times a week. According to OECD guideline 407, the oral route was chosen. The volume of administration was 10 mL/kg body weight, the volume of administration per animal was calculated by means of
the LIM system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for formulation analysis were scheduled to be taken from all dose groups at two
different time points for determination of concentrations at the Institute of Toxicology.
Analysis was performed using an HPLC method.

HPLC method no. TOX19-1/10 was developed and implemented at the Institute of Toxicology.
The raw data of the implementation are stored at the Institute of Toxicology. The limit of
detection (LOD) and the limit of quantification (LOQ) of this method was analyzed as 0.0003
and 0.001 mg/mL, respectively.

Reagents
Acetonitrile Lichrosolv® gradient grade (Merck Art. 1.00030)
Methanol Lichrosolv® gradient grade (Merck Art. 1.06007)
Water (Milli-Q).

Standard Solutions
Stock solutions of the test item in methanol were prepared for external standard
calibration. For each calibration curve, 50 mg of the test item was exactly weighed
into a 50 mL volumetric flask and approximately 40 mL methanol was added. Dissolution was
achieved by stirring the flask and ultrasonication (~ 5 min.). After that, the flask was filled to the
mark with methanol. Finally, six standard solutions were prepared by respective dilution of the
stock solutions with methanol to yield concentrations in the range from 0.010 to 0.250 mg/mL.

Analysis of Samples
For analysis, samples of each concentration were taken and provided to the analytical laboratory.
Depending on the dose group the samples were appropriately diluted with methanol and the
concentration of the test material was quantified using the calibration curve.

HPLC Conditions
Pump: VWR/Hitachi LaChrom Elite L-2130
Autosampler: VWR/Hitachi LaChrom Elite L-2200
Column oven: VWR/Hitachi LaChrom Elite L-2300
UV-VIS-Detector: VWR/Hitachi LaChrom Elite L-2420
Software: EZChrom Elite
Column: LiChroCart, LiChrospher 100 RP-18e, 125-4 mm, 5 μm
(Merck, Art. 50828)
Eluent: 1000 mL Acetonitrile
20 mL Water
Flow: 1.5 mL/min
Injection volume: 15 μL
Oven temperature: 22 °C
Wavelength: 228 nm
Washing solution: Water / acetonitrile (50:50, v/v)
Duration of treatment / exposure:
4 weeks exposure + 2 weeks recovery
Frequency of treatment:
daily for 28 days (7d/w)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)
Group 2: 100 mg/kg body weight: 10 (5m, 5f)
Group 3: 300 mg/kg body weight: 10 (5m, 5f)
Group 4: 1000 mg/kg body weight: 20 (10 (5m, 5f) test + 10 (5m, 5f) revovery)
Control animals:
yes, concurrent vehicle
Details on study design:
The test item is structurally similar to a chemical that was tested in a subacute repeat dose toxicity study for 28 days in rats at daily dose levels of
40, 200, and 1000 mg/kg. In this assay the high dose of 1000 mg/kg was not tolerated, animals had to be taken out of the study in moribund condition at the end of
the first week of administration. At 200 mg/kg some hematological and clinico-chemical parameters were changed. Additionally, increases of liver weight and atrophy of adrenal
cortex and inflammation were observed. The NOAEL was considered to be 40 mg/kg.

For the test item a 5-day dose range finding study in rats with oral gavage administration at dose levels of 100, 300 and 1000 mg/kg showed that 1000 mg/kg were tolerated
without any clinical signs. Mild to moderately toxic effects were expected with the test item at 1000 mg/kg with daily administration over a 28-day treatment period. 100 mg/kg and 300 mg/kg
were dose-levels to enable a dose-correlation of effects.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
SCHEDULE
Observations/Measurements Time schedule
Appearance and behavior daily
Mortality daily
Motor activity day 28
FOB predose (day 0), day 7, day 28
Body weight once a week
Food consumption once a week
Water consumption twice a week
Hematology week 5 +7
Clinical chemistry week 5 + 7
Urinalysis week 5 + 7

GENERAL CAGESIDE OBSERVATIONS
Mortality, the behavior and appearance of each animal were checked twice daily at working days and once at off days, preferably at the same time (s) each day. Symptoms were recorded with the LIM System

FOOD CONSUMPTION
Food consumption was determined once a week by weighing the food per cage which had not been consumed

WATER CONSUMPTION
Water consumption was determined twice a week by measuring the water per cage whichhad not been consumed. The parameter was recorded with the LIM-System.BODY WEIGHTSEach rat was weighed before treatment and thereafter once a week.

FUNCTIONAL OBSERVATIONAL BATTERY
females per group before the first exposure, a week thereafter (day 7), and in week 4 (day
28). On day 28, the functional observational battery was performed after the motor activity
measurement. Animal numbers and microchips were not identified for the laboratory staff
performing the functional observational battery. Therefore, the observers did not know to
which treatment group the rats belong.
The following parameters were examined in the cage, during removal of the cage, in the
observation arena and during handling: palpebral closure, ease of removal and handling
from cages, muscle tone, lacrimation, salivation, piloerection, fur appearance, mobility,
arousal, gait, approach response, touch response, click response, tail pinch response,
righting reflex, pupil response, raising, raising behavior, defecation and urination (number
of fecal boluses, feces consistency, number of urine pools, urine stain size), hind limb foot
splay, fore limb and hind limb grip strengths, internal body temperature, catalepsies,
posture, vocalization, convulsions, stereotypy, and any abnormalities.
The observed parameters were recorded in the LIM System.

MOTOR ACTIVITY
On day 28, one hour after administration, the rats (the numerical first 5 males and 5
females per group) were removed from their home cages and their motor activity was
recorded in special motor activity cages over 60 minutes at 5 minutes intervals. The
number of movements was evaluated by counting the number of interruptions of photo
beams (7 beams on the x-axis, 4 beams on the y-axis and 7 beams on the z-axis). These
parameters are called “counts” and given in numbers. The system was also calculating the
“on-time” (minutes), “off-time” (minutes), the “total distance” the animal was moving
(meter), “rearing” (number), and “rearing time” (minutes). The data was transferred into
the LIM System. Assignment of the rats to the individual measurements followed a
randomization schedule. On each measuring day, measurements were conducted
simultaneously in 10 rats.

LABORATORY TESTS
In weeks 5 and 7 blood was taken under inhalation anesthesia from the rats listed below.
Except for the animals that were kept for recovery, blood was taken before necropsy in
week 5. The blood samples were divided for hematological and clinico-chemical
examinations. Before blood sampling the animals were kept in metabolism cages for the
collection of urine for approximately 18 hours without food.
Sacrifice and pathology:
NECROPSY
Sacrifice:
After 4 Weeks
After 6 Weeks (Recovery)
At the end of the study the rats were anesthetized by a carbon dioxide air mixture andexsanguinated by opening the abdominal vessels. The rats were necropsied and examinedfor gross pathological alterations.
ADRL Adrenal
N SC Nerve, sciatic
AORT Aorta
OVAR Ovary
BONE Bone
OVID Oviduct
BRN Brain
PANC Pancreas
DUOD Intestine, small, duodenum
PARA Parathyroid
ESOP Esophagus
PEYP Peyer's patches
EPID Epididymis
PITU Pituitary
EYE Eye
PROS Prostate
HRT Heart
SALI Salivary glandI
CE Intestine, large, cecum
SEMV Seminal vesicleI
CO Intestine, large, colon
SKIN SkinI RE Intestine, large, rectum
SPIN Spinal cordI
LEU Intestine, small, ileum
SPLN Spleen
JEJU Intestine, small, jejunum
STOM Stomach
KIDY Kidney
TEST Testis
LIVR Liver
THYM Thymus
LUNG Lung
THYR ThyroidM
SK Muscle, skeletal
TRAC Trachea
MAND Lymph node, mandibular
UR Ureter
MAMY Mammary gland
URIN Urinary bladder
MARR Bone marrow
UTER Uterus
MESE Lymph node, mesenteric
VAGI Vagina
NOP Nerve, optic
REPRO, Reproductive organs

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
Terminal body weight (after exsanguination)
HeartLiverKidneys (together)SpleenThymusTestes (together)
ProstateOvaries (together)
UterusAdrenals (together after fixation)
Thyroids with parathyroids (together after fixation)Brain (cerebrum, cerebellum, medulla oblongata afterfixation)Epididymides (together)Seminal vesiclesHISTOTECHNIQUEFor the main kill animals organs and tissues were fixed, histotechnically processed andexamined as listed below.Adrenal (2)AortaBone with knee joint (os femoris)Bone with bone marrow (sternum, femur)Brain (cerebrum, cerebellum, brain stem)EsophagusEye (2)HeartIntestine, large Cecum Colon RectumIntestine, small Duodenum Jejunum IleumKidney (2)LarynxLiver (left lateral and right medial lobe)Lung (with mainstem bronchi)Lymph nodes mandibular (2) mesentericMammary gland (inguinal)Micro transponderMuscle, skeletal (thigh)Nasal turbinatesNerve, optic (2)Nerve, sciaticPancreasParathyroid (2)Peyer’s PatchesPituitaryReproductive organs, femaleOvary (2) Oviduct (2)Uterus (cornu/corpus/cervix) VaginaReproductive organs, male Epididymis (2) ProstateSeminal vesicleTestis (2)Salivary gland (2)(submandibular, parotid, sublingual)Skin (inguinal)Spinal cord (cervical, thoracal, lumbal)SpleenStomach (proventricular, fundic, pyloric)ThymusThyroid (2)TongueTracheaUreter (2)Urinary bladderZymbal's gland (2)All tissues showing abnormality

HISTOPATHOLOGY

Slides of all organs and tissues listed above which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. As histomorphologic changes were observed in the liver of high-dose animals, the livers of the animals of the low- and mid-dose group were also examined.
Statistics:
All parameters were analyzed separately for each sex and time. To take the number of dosegroups into account all the test procedures used maintain a multiple significance level ofa= 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
@1000, and 300 mg/kg bw
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain were significantly decreased in group 4 males and
females (1000 mg/kg) compared to control resulting in approximately 8 % less body
weight and 26 and 38% less weight gain in males and females, respectively. The other
treatment groups did not show significant effects on body weight.
During the recovery period the body weights of group 4 animals recovered partially, the
rats had about 5% less body weight on day 42 than the concurrent controls, the weight gain
was at approximately 80 – 85 % compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Some significant differences of food consumption from control were observed in various
treatment groups. However, the changes were mild, increases and decreases seen and the
parameter is evaluated per cage, not individually. Therefore, the changes are not
considered treatment-related.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption was slightly increased in male group 2 animals (100 mg/kg) versus
control at several time points (days 3, 10), in male group 3 animals (300 mg/kg) versus
control at several time points (days 10, 17, 21 and 24) and in male group 4 animals (1000
mg/kg) versus control at several time points (days 31 and 35).
For female animals the water consumption was slightly increased in group 2 animals (100
mg/kg) versus control at several time points (days 7, 10, 17, 21 and 24) and in female
group 4 animals (1000 mg/kg) versus control at one time point (day 24). No changes were
seen in group 3 female animals (300 mg/kg).
However, no dose-dependent effect could be seen, therefore the changes are not considered
treatment-related.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological evaluation at the end of the treatment period showed slight deviations of red
blood cell parameters (decrease of haemoglobin) in females at the end of the treatment
period and in males of group 4 (1000 mg/kg) at the end of the recovery period. The
changes seemed to be of a compensatory nature based on an increase of reticulocytes.
Leukocytes were clearly decreased in females group 4 (1000 mg/kg) only, this change was
fully reversible at the end of the recovery period. The slight decrease of prothrombin time
at the end of the recovery period in group 4 females (1000 mg/kg) and the slight increase
of platelets in group 2 females (100 mg/kg) in test week 5 are considered to be of no
pathological relevance due to the less degree and inconsistency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry evaluation showed that at the end of the treatment period, triglycerides
were slightly but significantly decreased in all treated males. Cholesterol was decreased in
group 4 males (1000 mg/kg), only. Albumin was slightly decreased in group 2 males (100
mg/kg) and group 4 females (1000 mg/kg). A/G ratio was slightly decreased in group 4
animals (1000 mg/kg). All changes were within the internal laboratory range except for
cholesterol in group 4 males (1000 mg/kg). Decreases of the altered parameters
cholesterol, triglyceride, albumin and albumin/globulin ratio could be caused by an
increased liver metabolism. At the end of the recovery period cholesterol, triglycerides,
albumin, and A/G ratio had fully recovered.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis showed no relevant deviations when compared to the concurrent controls. A
slight decrease of specific gravity evident in group 2 females (100 mg/kg) is considered to
be of no pathological relevance due to the low degree, and missing further alteration of the
kidney.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
@1000 , and 300 mg/kg bw
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
During necropsy at the end of the treatment period, a dose-dependent liver weight increase
correlating with centrilobular hypertrophy in female animals at all dose levels was
observed. This is interpreted to be adaptive and to represent metabolic activation, a
common phenomenon after administration of a xenobiotic. Females were more susceptible
than males, which is also reflected in the body weight decrease at 1000 mg/kg in this sex.
The liver weight increase showed a tendency towards reversibility after the 2 week
recovery period. Centrilobular hypertrophy was not observed in recovery animals and was
therefore completely reversible.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At gross pathology only spontaneous findings were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathology, a mild centrilobular liver cell hypertrophy was observed in female rats of all
dose groups. At 100 mg/kg four rats were affected, at 300 and 1000 mg/kg all females exhibited
centrilobular hypertrophy. This finding correlates with the observed liver weight increase in
females. In some rats of the 300 and 1000 mg/kg dose group there was also a minimal increase
of centrilobular single cell necrosis. This finding is of questionable toxicological relevance as it
was only minimal in severity and male rats were also affected which did not reveal liver weight
changes or centrilobular hypertrophy.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
behaviour (functional findings)

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver

Applicant's summary and conclusion

Conclusions:
A NOEL (no observed effect level) could not be established in the present study. 300 mg/kg bw/d are considered to be the NOAEL (no adverse effect level).