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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-16 to 2016-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (main study)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

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
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Chemical Name: Carbamic acid, N-(3-(triethoxysilyl)propyl)2-(ethyl-(4-nitrosophenyl) amino) ethyl ester
- Name (as cited in the report): SAT 140016
- Batch No.: 3801-75
- Physical State: powder
- Colour: green
- Active Components / Purity: 87.7 area % (HPLC)
- Date of Analytical Report: 13 January 2015
- Storage Conditions: room temperature, protected from light and humidity
- Expiry Date: 09 January 2016
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS
- Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Sex: male and female; the female animals were non-pregnant and nulliparous.
- Age at the start of the treatment period: males: 7-8 weeks old, females: 7-8 weeks old
- Body weight at the time of allocation of the animals to the experimental groups:
males: 186.1 – 216.8 g (mean: 202.97 g, ± 20% = 162.38 – 243.56 g)
females: 137.9 – 166.7 g (mean: 153.89 g, ± 20% = 123.11 - 184.67 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

PREPARATION
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
Before the first day of administration all animals used for the study were weighed and randomized to the experimental groups to minimize variation in body weight throughout the groups

HOUSING AND FEEDING CONDITIONS:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1239)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre
bedding (lot no. 02102141114).
- Certificates of food, water and bedding analyses are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
EXPERIMENTAL GROUPS AND DOSES
According to the results of a previous dose range finding study (BSL Project 141669) and in consultation with the sponsor the following doses
were selected for the 3 dose groups (LD = low dose, MD = mid dose, HD = high dose):

C 0 mg/kg bw
LD 100 mg/kg bw (referred to active components)
MD 300 mg/kg bw (referred to active components)
HD 1000 mg/kg bw (referred to active components)

The animals were treated daily with the test article or vehicle for a period of 28 days and day 1 represents the first day of treatment.
5 animals per sex per group were subjected to necropsy one day after the last administration (end of treatment period).
Five animals per sex of the C and of the HD group were allowed to recover for 14 days after the last administration of the test article
and subjected to necropsy (end of recovery period).
The highest dose level was chosen with the aim of inducing toxicity without excessive mortality or severe suffering. Thereafter, a descending
sequence of dose levels (MD and LD) was selected to demonstrate any dose related response and to determine a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received a similar volume of the vehicle
as used for the high dose group.

ADMINISTRATION
The test article formulation and vehicle were administered twice daily to the animals by oral gavage to 3 dose groups using
disposable polyurethane feeding tubes (13 ga x 88 mm; or 15 ga x 78 mm, Instech Laboratories Inc.) with approx. 3 hours in between.
The administered volume for all groups was 4 mL/kg body weight using the most recently measured body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the concentration of test article in dosing formulations, samples of at least 5 mL were retained from all groups
once weekly during the treatment period (in total 16 samples).
In the first and fourth week of treatment, samples from the top, middle and bottom of the freshly prepared high and low dose formulations
were tested for homogeneity (total 12 samples).
Each sample was prepared in duplicate (sample A, sample B, each of at least 5 mL). Sample A was stored at room temperature and transferred
on the same day to the analytical department (Test Site 3). Sample B served as safeguard and was stored at -15 to -35 °C. The B samples were
retained at BSL (conditions as above-mentioned) and will be discarded after the completion of the final study report. Sample were analysed
according to the analytical phase plan of the study (Eurofins Study No.: 141671). The results were reported in the appendix of the final report.
Duration of treatment / exposure:
The animals were treated daily with the test article or vehicle for a period of 28 days and day 1 represents the first day of treatment.
Frequency of treatment:
The animals were treated twice daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg bw, 300 mg/kg bw and 1000 mg/kg bw (referred to active components)
Basis:
actual ingested
No. of animals per sex per dose:
60 animals (30 males and 30 females) were included in the study and divided into 4 groups of either 5 males and 5 females for the LD
and MD group or 10 males and 10 females for the control and HD groups. 5 males and 5 females from the control and HD groups served
for analysis after the recovery phase.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: random
- Section schedule rationale : alle male animals together and all female animals together

Examinations

Observations and examinations performed and frequency:
BODY WEIGHT AND FOOD CONSUMPTION
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly
during the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.

CLINICAL OBSERVATIONS
All animals were observed for clinical signs during the entire treatment period of 28 days.
The recovery animals were observed for an additional period of 14 days following the last test article or vehicle administration.
General clinical observations were made at least once a day, at the same time each day and at the anticipated peak effect after dosing.
The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends
and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of
the treatment period as well as at the end of the recovery period in the recovery animals.

FUNCTIONAL OBSERVATIONS BATTERY
Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period multiple
detailed behavioural observations were made outside the home cage using a functional observational battery (FOB) of tests.
These tests were conducted on all animals.

EXAMINATION OF DISCOLOURED SAW FIBRE BEDDING
Red discoloration of the saw fiber bedding was observed in cages of animals from the HD group on study day 6. To examine the reason for
the discoloration all animals of the HD group (males and females) were transferred to metabolic cages on study day 9 or 10 (for male animals
of the recovery group) for approx. 5 ½ hours in order to collect urine and faeces. During the housing in metabolic cages food and water were
available ad libitium. Faeces samples were frozen and stored at -15 to -35 °C. Urine samples were examined to detect any occurrence of blood
using qualitative indicators (e.g. Heiland Urine Stripes URI 10SL). Urinalysis was performed on urine samples collected including observation
of urine colour / appearance. Furthermore, neat saw fibre bedding was moistened with urine and test article as follows:

1 saw fibre bedding urine from HD animals -
2 saw fibre bedding SAT 140016 in corn oil* -
3 saw fibre bedding urine from HD animals SAT 140016 in corn oil*
* = same concentration as applied for administration of HD animals
All samples from experiment 1 to 3 were placed in petri dishes and incubated at room temperature for 9 days.
Discoloration was observed and recorded.

EXAMINATION OF FERTILITY PARAMETERS
Daily over a period of 8 days, the estrous cycle of all female animals was examined during fourth week of exposure.
At the scheduled necropsy at the end of the treatment phase (one day after last administration) and at the end of the recovery period,
left epididymis, left testis and left vas deferens were collected for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in male animals using Hamilton Thorne Sperm Analyser
(TOX IVOS Version 13.0). For evaluation of morphology, sperm from left vas deferens was transferred to 0.1% bovine serum albumin solution.
For staining two drops of 1% aqueous Eosin-Y solution was mixed with six drops of the sperm-suspension. The stained sperm suspension
was used to prepare smears on slides. The slides were dried and dipped into 0.1% acetic acid for approximately 30 seconds to intensify the coloring.
Sperm morphology examinations were evaluated at AnaPath GmbH in male animals of all groups sacrificed at the end of the treatment period.
Sacrifice and pathology:
HAEMATOLOGY
Haematological parameters were measured at the end of the treatment and recovery period as part of the sacrifice of the respective animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells

BLOOD COAGULATION
Coagulation parameters were evaluated at the end of the treatment and recovery period as part of the sacrifice of the respective animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL BIOCHEMISTRY
Parameters of clinical biochemistry were evaluated at the end of the treatment and recovery period prior to or as part of the sacrifice of
the respective animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT),
alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol),
glucose (Gluc), sodium (Na), potassium (K)
Additionally, at necropsy serum samples of all animals were retained at the end of the treatment and recovery period and stored at -15 to -35° C.

URINALYSIS
A urinalysis was performed with samples collected from all animals at the time of necrospsy.
Additionally, urine colour / appearance were recorded.
The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL):
specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery),
leukocytes (Leu)

PATHOLOGY
One day after the last administration (study day 29) all surviving animals of the treatment period and 2 weeks after the last administration
all surviving animals of the recovery period (study day 43) were sacrificed using anesthesia (ketamine, Pharmanovo Arzneimittel, Lot No: 24863,
expiry date: 10/2015 and xylazin, Rebopharm, Lot No. 400260/1, expiry date: 01/2017) and subjected to a detailed gross necropsy which
includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGHT
The wet weight of the following organs of all sacrificed animals was recorded in the procedure of necropsy. Paired organs were weighed together.
Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded:
liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides, brain, prostate,
seminal vesicles and coagulating glands, pituitary gland, ovaries, heart

The following tissues from all animals were preserved in 4% neutral-buffered formaldehyde with the exception of eyes, testes and epididymides
which were fixed in Modified Davidson’s fixative for approximately 24 hours before being transferred to 70% ethanol:
brain (cerebrum, cerebellum and pons), spinal cord, eye, liver, kidneys, adrenal glands, stomach, small and large intestines
(including Peyer´s patches), thymus, thyroid glands, spleen, lung and trachea, mammary glands, skin, heart, ovaries (females),
uterus with cervix (females), vagina (females), testes (males), epididymides (males), prostate and seminal vesicles with coagulating glands as a whole
(males), urinary bladder, lymphnodes (mesentric and axillary), peripheral nerve (e.g. sciatic nerve) with skeletal muscle, sternum with bone marrow,
pituitary gland, oesophagus, gross lesions
All animals found dead and/or intercurrently euthanised for animal welfare reasons were also subjected to a gross necropsy and the
organs preserved for a possible histopathological examination at the discretion of the Study Director.

HISTOPATHOLOGY
The afore-listed organs (Table 8) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining
for the animals of the groups 1 and 4 sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned
day of sacrifice. In addition, liver and kidneys were evaluated histopathologically in all animals of LD, MD and recovery groups. Any gross lesion
macroscopically identified was examined microscopically.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of
additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified
contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The Study phases from test site 1
and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1]
(Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to
the corresponding SOP’s of the test sites. The Principal Investigator for histopathological evaluation provided the histopathology results to the
Study Director by e-mail and provided a pathology phase report to the Study Director upon the completion of the study.

Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals
using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed w
ith a Student’s t-Test. These statistics were performed using GraphPad Prism V.6.01 software or IDBS E-WorkBook 9.4.0 software
(p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
One of 10 male (animal no 19) and 1/10 female animals (no 48) of the high dose group died during the treatment period of the study.
Male animal no. 19 was found dead on study day 25 and showed mild clinical signs including moving the bedding and salivation during the
treatment period and prior to. Female no. 48 was found dead on study day 27. Although a cause of death could not be determined by
histopathological examination for neither of the two animals, animal no. 48 showed respiratory sounds and dyspnoe a day prior to death and
a dark discoloration on the oesophagus at necropsy was observed, suggesting a local effect caused by gavaging.
One of 10 female control animals (no. 52) died during the treatment period of the study (study day 8 and 5 minutes after administration).
No clinical symptoms were observed in this animal prior to death. Histopathological evaluation of the organs led to the diagnosis of fat
embolism as a contributing factor of death. However, a gavage error could not be excluded.

CLINICAL OBSERVATIONS
Clinical signs including moving the bedding and salivation were observed in all animals treated with SAT 140016, starting at the end of the first
week or beginning of the second week of administration (LD and MD groups) or as early as study day 2 (HD group). While these symptoms were
observed only occasionally at the low dose group, they were noted in all animals of the mid and high dose groups throughout the treatment
period, from early in the first week onwards. Moving the bedding and salivation closely coincided with administration of the test article and
was not seen during the recovery period. For all animals on study day 9 or 10 (for male recovery animals) a yellow discoloration of the bedding
was observed in the low dose group and a red discoloration was seen at the medium and high dose level. To investigate the cause of this effect,
additional investigations were performed. On study day 15, male no. 30 of the high dose group showed marked respiratory sounds,
half-closed eyes and piloerection. The animal recovered the next day with the exception of only a slight piloerection. A slight lesion might
have been caused by the gavaging cannula which would explain the abovementioned clinical observations. However, in the trachea of that
animal no histological findings were recorded. Occasional clinical signs observed on single or consecutive days including slightly increased
spontaneous activity and slight respiratory sounds or slight piloerection are not assumed to be a systemic effect of SAT 140016.
No other clinical observations were noted during the weekly detailed examination and no other significant changes or differences between the
groups were identified. There were no ophthalmoscopic findings in any of the animals of this study.

FUNCTIONAL OBSERVATION BATTERY
No relevant effects of SAT 140016 were observed on any of the parameters of the functional observation battery at the end of the
treatment or recovery period. There were no biologically relevant differences in body temperature between the groups.

BODY WEIGHT DEVELOPMENT
In both males and females the body weight increased with the progress of the study. The mean daily weight gain was slightly but statistically
significantly higher in the female high dose group (1.40 g/day) compared to the control (1.05 g/day). This effect was not observed in male animals.
Moreover the absolute mean body weight was in the range of 94.6 % to 102.0 % in males and 100.9 % to 106.3 % in females, which is within the
normal range of historical control data. Therefore the slightly higher mean weight gain in high dose females is considered not to be toxicologically
relevant.

FOOD CONSUMPTION
No effect of SAT 140016 on food consumption was noted in this study. Throughout the treatment and recovery period there were no
differences between treatment groups and the control group of this study.

FERTILITY PARAMETERS
At the end of the treatment period there was no effect of SAT 140016 on fertility parameters measurements in this study. Sperm motility
parameters (rate of motile, static and rapidly moving sperms) were not significantly different between test article treated and control animals.
There was also no difference in sperm count between SAT 140016-treated and control animals.
At the end of the treatment period, the length of the estrous cycle was either 4 or 5 weeks in control animals (9/9). This deviated, showing
irregularities in estrous cycle in 3/5 animals of the low dose group, 1/5 animals of the mid dose group and 3/9 animals of the high dose group.
Because of the high variability and the missing dose dependenteffect of SAT 140016, changes in the duration of estrous cycle were considered
not to be test article related. In addition, there were no histopathological correlates at the high-dose group females.

HAEMATOLOGY AND BLOOD COAGULATION PARAMETERS
Daily oral treatment with SAT 140016 had no adverse effect on haematology or blood coagulation parameters.
At the end of the treatment period haematological parameters were in the normal range of historical control data in most animals.
Platelet count, WBC and red blood cell parameters (RBC, Hb and Hct) were low in male animal no. 6 of the low dose group compared to the control.
This finding was considered incidental and not test article related, since no changes in these parameters was observed in mid or high dose
group animals. A tendency towards higher differential count of neutrophils and a lower differential count of lymphocytes was observed in
2/5 female animals at 1000 mg/kg/d (animals no. 49 and 50). Animal no. 49 also displayed a higher differential count of eosinophils.
These changes may be related to a possible infectious reaction and not considered adverse or test article related.
All other haematological parameters were within the normal range of variation with no statistically significant differences between dose groups
and control group. There was no effect on coagulation parameters measured at the end of the treatment period.
At the end of the recovery period there were no biologically relevant differences in haematological parameters or coagulation parameters between
SAT 140016-treated animals and control animals.

CLINICAL BIOCHEMISTRY
No effect of SAT 140016 on clinical biochemistry parameters of the animals was observed at the end of the treatment period.
At the end of the treatment period female animal no. 47 showed increased serum level of Crea and Urea serum levels
(182 µmol/L and 14.54 mmol/L, respectively), compared to controls (mean of 31 µmol/L and 6 mmol/L, respectively).
This finding correlated with minimal or slight basophilia and vacuolation of the kidney. Similarly, at the end of the recovery period, Crea and
Urea serum levels of male animal no. 28 of the high dose group were elevated (110 µmol/L and 12.26 mmol/L, respectively) compared to
controls (mean of 25 µmol/L and 6.5 mmol/L, respectively). This change was not associated with pathological signs of the renal system and
was not considered adverse or related to SAT 140016 administration. All other clinical chemistry parameters were within the normal range
of variation for this strain and there were no statistically significant differences noted between test article treated and control groups.

URINALYSIS
SAT 140016 had no effect on urinary parameters determined at the end of the treatment period or at the end of the recovery period.
Occasionally, high count of erythrocytes was observed not only in the urine of SAT 140016-treated animals but also in the control group
at the end of the treatment and recovery period. Therefore this finding was considered not adverse or test article related.

EXAMINATION OF DISCOLOURED SAW FIBRE BEDDING
Red discoloration of the saw fiber bedding was observed in cages of animals from the HD group on study day 6. To examine the reason
for the discoloration urine from all animals of the HD group (males and females) was collected on study day 9 or 10. Urinalysis was performed
and, in addition, a reaction of saw fiber bedding in combination with the urine, the test article or both was assessed by visual investigation.
Urinalysis of animals of the high dose group performed on study day 9 or 10 showed that urine contained no detectable numbers of erythrocytes.
On the other hand, ketone bodies were found in all female and 2/10 male animals of this group. Besides, there were no remarkable findings in
urinary parameters of these animals. The combination of urine samples and saw fibre bedding or test article formulation and saw fibre
bedding or a combination of the three, supported the hypothesis that the discoloration observed in the bedding of the animals of the high
dose group was caused by the test article. A mixture of saw fibre bedding and test article formulation resulted in a yellow colour. Urine of high
dose animals alone or in combination with formulation of the high dose group led to a yellow-green or brown-red discoloration that turned
into green-brown over the period of 10 days.

PATHOLOGY
Macroscopic changes observed in high-dose male no. 19 found dead on day 25 included dark discoloration and fluid distension in the lungs
and autolysis of brain and pituitary gland. Changes in the lung indicate a gavage error which is not test article related. Macroscopic changes
observed in high-dose female no. 48 found dead on day 25 included dark discoloration of the esophagus, soft kidneys, brain and pituitary gland.
For both animals the cause of death could not be established but was unlikely related to the test article.
Macroscopic changes observed in control female no. 52 that died 5 minutes after administration on day 8 included dark discoloration of the
stomach, jejunum and lungs. In histopathology this animal was considered to be died by fat embolism.
No other histopathological changes could be detected in the remaining animals at necropsy that could be attributed to treatment with SAT 140016.
Incidental findings at the end of the treatment period included yellow focus on the epididymis (LD male no 12) which was identified in histology
as sperm granuloma, dilated pelvis in the kidney (LD male no 7) which is confirmed histologically, a dark focus on the thymus (control male no 5)
which was identified as congestion and fluid distensions (MD female no 45, HD females no 50 and 48) of the uterus which were found in histology
to be related to cyclic change. Single or occasional findings observed at the end of the recovery period were yellow focus on the testes
(control male no 22) with no histological correlate and on the epididymis (control male no 24) histologically identified as perm granuloma.
Moreover, fluid distension of the uterus (control female no 53 and HD female no 60) which were found in histology to be related to cyclic change.
These changes were not dose-dependent, were also observed in control animals and therefore not considered test item related.

ORGAN WEIGHT
The liver weight of female animals was statistically significant slightly higher at 1000 mg/kg/d and very slightly higher at 300 mg/kg/d compared
to controls, recorded at the end of the treatment period. This increased liver weight was correlated with minimal hepatocellular hypertrophy in
3 of 5 HD females. The related histopathological finding was a minimal hepatocellular hypertrophy in several HD females.
This finding was not associated to any further degenerative or inflammatory lesion, hence is deemed to be adaptive but not adverse in nature.
No histological correlation was observed in the 300 mg/kg/d females. Histopathological analysis deems that this findings is adaptive and not
adverse. At the end of the treatment period, increased absolute and relative to brain kidney weight was recorded in 1000 mg/kg/d group female
(16 and 17 %, respectively) compared to control. Increase in kidney weights was associated with minor increased tubular epithelial vacuolation
whereby the cortical regions (including the corticomedullary junction) were mainly affected in females administered 1000 mg/kg. No difference
in kidney weight or histopathological findings were noted at the end of the recovery period or in males.
A statistically significant higher adrenals weight (24% relative to body weight) in 1000 mg/kg/d group males compared to controls was observed
at the end of the treatment period. Increased adrenals weights was not associated with histopathological findings and therefore not considered
to be toxicologically relevant. No changes were observed in the high-dose group females.
Slight increased (p< 0.05) heart weight (8% relative to body weight) of male animals of the high dose group was observed at the end of the
recovery period compare to control animals.
Dose-dependent increased in absolute and relative uterus weight, where non-significant differences were found at 300 and 1000 mg/kg/d
(up to 50 % above controls). The changes were not considered toxicologically relevant changes because the variation in uterine weight was large,
within historical control values, and not statistically significant.
Weight of ovaries was slightly but not significantly increased at the low and high dose levels – but not at 300 mg/kg/d. At the end of recovery
period ovary weight was statistically significantly higher in animals of the high dose group than in the respective controls (approx. 39 %).
Slight and non-statistical 30% increase in Thyroid weight was observed in high-dose male group at the end of the recovery period only.
Statistically higher testis weight of male animals of the high dose group, was observed at the end of the treatment period (16 % higher than controls).
With the exception of kidneys of female animals at 1000 mg/kg/d, none of the above-mentioned weight changes was associated with
histopathological findings and is therefore, not considered toxicologically relevant. Besides, no considerable differences in organ weight
were observed between dose and control group of this study.

HISTOPATHOLOGY
In females administered 1000 mg/kg, there was a minimal centrilobular hepatocellular In females administered 1000 mg/kg, there was a
minimal centrilobular hepatocellular hypertrophy in 3 out of 5 animals as well as increased in liver weights. The former finding was not associated
with any further degenerative or inflammatory lesion, hence was deemed to be adaptive (suggestive for enzyme induction) but it is not adverse in
nature. Moreover, this finding in liver reversed during the treatment-free period.
There was a minor increased tubular epithelial vacuolation in 5 out of 5 females at 1000 mg/kg. Tubular vacuolation was also seen in one control
female but not in low and mid dose females. The renal findings were reversible with no effects observed after the treatment-free period.
Renal tubular vacuolation is reported to be a transient finding under the use of corn oil as vehicle (Haschek, Wallig, and Rousseaux, 2010).
Therefore, this finding is not considered adverse in nature. There were no sperm abnormalities recorded either by staging or smear morphology.

DOSE FORMULATION ANALYSIS
The mean recoveries observed in the low dose (LD), mid dose (MD) and high dose (HD) groups were 100%, 98% and 94% of the nominal
concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured mean concentrations were within
acceptance criteria of 20%. The mean recovery observed for LD dose group was 94% and for HD dose group 86% and 102% of the nominal value.
The coefficients of variation of the different sampling locations (top, middle, bottom) were 20% and 19% in LD dose group, and 8% and 1% in HD
dose group. All samples were homogenous, as COV was below or equal 20%.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Concerning:     Examination of Fertility Parameters (study plan, p. 16)

Study Plan:

For evaluation of morphology, sperm from left vas deferens will be transferred to 0.1% bovine serum albumin solution. For staining two drops of 1% aqueous Eosin-Y solution will be mixed with six drops of the sperm-suspension. The stained sperm suspension is used to prepare smears on slides. After complete drying the slides are dipped into 0.1% acetic acid for approximately 30 seconds to intensify the coloring. Sperm morphology examinations will be evaluated at AnaPath GmbH in male animals of the groups 1 and 4 sacrificed at the end of the treatment period. This analysis will be extended to male animals of all other dosage groups and male animals of recovery group for treatment-related changes that are observed in the high dose group.

Report:

For evaluation of morphology, sperm from left vas deferens was transferred to 0.1% bovine serum albumin solution. For staining two drops of 1% aqueous Eosin-Y solution was mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. The slides were dried and dipped into 0.1% acetic acid for approximately 30 seconds to intensify the coloring. Sperm morphology examinations were evaluated at AnaPath GmbH in male animals of all groups sacrificed at the end of the treatment period.

Reason:

Inadvertently, sperm morphology analysis was extended to male animals of LD and MD group, although no treatment-related changes were observed in the high dose group. However, that data is reported and gives a better understanding of test article toxicity.

Applicant's summary and conclusion

Conclusions:
In this 28-day repeated dose oral toxicity study, SAT 140016 was administered to 10 Wistar rats (5 males and 5 females) per dose levels of
100, 300, and 1000 mg/kg body weight/day, followed by a recovery period of 14 days (5 males and 5 females in control and high dose).
Slight clinical signs observed after administration were assumed to be related to a local effect caused by oral gavaging of the test article
formulation, but are not considered signs of systemic toxicity.
In 1000 mg/kg group females, there was slight increase in liver weights (123% absolute mean weight compared to control) accompanied by
minimal hepatocellular hypertrophy. This finding was not associated with any further degenerative or inflammatory lesion and hence is deemed
to be adaptive but not adverse in nature. In addition, increased kidney weights in females administered 1000 mg/kg was associated with minor
increase in tubular epithelial vacuolation whereby the cortical regions (including the corticomedullary junction) were mainly affected.
The renal finding was reversible and not considered adverse in nature.
Overall, no signs of systemic toxicity were observed up to a dose level of 1000 mg/kg/d and therefore the NOAEL for toxicity in this study
was determined to be 1000 mg/kg/day for males and females.
The NOAEL for fertility parameters (functional or morphological) was 1000 mg/kg/d for both males and females in this study.
Executive summary:

Objective of the Study

The aim of this study was to evaluate the possible health hazards which could arise from repeated oral administration of SAT 140016 to rats

Summary Method

The test article was administered daily to 3 groups of Wistar rats (5 males and 5 females per group) at doses of 100, 300 and 1000 mg/kg/day

for a treatment period of 28 days. An additional group of animals served as control group and animals were handled identically as the test animals but received corn oil, the vehicle used in this study. Animals were exposed to the test article or control formulation by oral gavage twice daily at an interval of 3 hours. The test article formulation was prepared freshly on each day of administration and dose volume was adjusted individually based on weekly body weight measurements. Animals were observed each day for signs of toxicity during the administration period. Animals that died prior to scheduled necropsy were examined macroscopically. Animals that survived to the scheduled necropsy were euthanized at the conclusion of the test, and observed macroscopically. 

Body weight and food consumption were measured weekly. Clinical pathology was evaluated at the end of the treatment period. In addition, organ weights (wet weight) were measured and tissues collected from the high dose and control animals were subjected to histopathological evaluation. At the end of the treatment period estrous cycle and sperm count and sperm motility were analyzed in the respective gender.

To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for an additional period of 14 days following the last administration of the test article or vehicle. Liver and kidneys of all animals (treatment and recovery groups) were evaluated histopathologically

Summary Results

On the basis of the present study, the 28-Day Repeated Dose Oral Toxicity study with SAT 140016 in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg body weight/day the following conclusions can be made:

All animals survived to the scheduled necropsy with the exception of one out of ten male and one out of ten female animals of the high dose group which died on day 25 or 27, respectively.

In addition, one of ten females of the control animals died on day 8 during the treatment period of the study. A contributing factor to death of the control animal was fat embolism possibly caused by a gavage error. The cause of death for the remaining two animals (one male and one female) of the high dose group is likely attributed to oral gavage error. This is suggested by respiratory sounds and dyspnoe noted a day prior to death. Necropsy of these animals was unremarkable and, therefore, a relationship of the test article to death is unlikely.

Mild clinical signs were noted immediately following test article administration consisting of moving the bedding and salivation. Functional observation battery revealed no effect of SAT 140016 on neurological parameters analysed at the end of the treatment period. No alteration in body temperature was found and there were no ophthalmoscopical findings in this study. SAT 140016 had no effect on body weight, body weight gain or food consumption.

Fertility parameters analysed at the end of the 28-day treatment period revealed no effects of SAT 140016. This was supported by the histopathological evaluation of reproductive organs and of sperm staging and sperm morphology, where no test article-related abnormalities were observed.

At the end of the treatment and recovery period no effects of SAT 140016 on haematological, coagulation parameters, or clinical biochemistry markers were observed. SAT 140016 had no effect on urinary parameters determined at the end of the treatment period or at the end of the recovery period.

At the end of the treatment or recovery periods, no macroscopic abnormalities were detected that could be attributed to treatment with SAT 140016.

Slightly higher kidney weight (mean values compared to control: 116% absolute, 117% relative to brain, 114% relative to body weight) was observed in females at a dose of 1000 mg/kg/d at the end of the treatment period which completely recovered at the end of the 14-day recovery period. Increased kidney weight in females was associated with an increased incidence of tubular epithelial vacuolation, mainly the cortical regions (including the corticomedullary junction) were affected. Tubular vacuolation was also seen in one control female but not in low and mid dose females. The renal findings were reversible with no effects observed after the treatment-free period. Renal tubular vacuolation is reported to be a transient finding under the use of corn oil as vehicle (Haschek, Wallig, and Rousseaux, 2010). Therefore, this finding is not considered adverse in nature. Male animals of this group were unaffected by the test article.

Liver weight was slightly but significantly higher in females at 1000 mg/kg/d (mean values compared to control: 123% absolute, 127% relative to brain, 120% relative to body weight) and very slightly higher in females at 300 mg/kg/d (mean values compared to control: 114% absolute, 114% relative to brain, 110% relative to body weight). No changes in liver weights were detected in male at any dose groups.

In females administered 1000 mg/kg/d,a minimal centrilobular hepatocellular hypertrophy was observed at minor degrees of severity. Hepatocellular hypertrophy was not observed in females at 300 mg/kg/d or in males at any dose groups examined. This finding was not associated with any further degenerative or inflammatory lesion, hence is deemed to be adaptive (suggestive for enzyme induction) and not adverse in nature. Moreover, this finding in liver reversed during the treatment-free period.

There were no sperm abnormalities recorded either by staging or smear morphology.

Formulation analysis showed that the acceptance criteria was met, i.e. the mean recoveries were in the range of 94% to 100% of the nominal concentration. All samples were homogenous, as COV was below or equal 20%. 


Conclusion

In this 28-day repeated dose oral toxicity study, SAT 140016 was administered to 10 Wistar rats (5 males and 5 females) per dose levels of

100, 300, and 1000 mg/kgbody weight/day, followed by a recovery period of 14 days (5 males and 5 females in control and high dose).

Slight clinical signs observed after administration were assumed to be related to a local effect caused by oral gavaging of the test article formulation, but are not considered signs of systemic toxicity.

In 1000 mg/kg group females, there was slight increase in liver weights (123% absolute mean weight compared to control) accompanied by minimal hepatocellular hypertrophy. This finding was not associated with any further degenerative or inflammatory lesion and hence is deemed to be adaptive but not adverse in nature.

In addition, increased kidney weights in females administered 1000 mg/kg was associated with minor increase in tubular epithelial vacuolation whereby the cortical regions (including the corticomedullary junction) were mainly affected. The renal finding was reversible and not considered adverse in nature.

Overall, no signs of systemic toxicity were observed up to a dose level of 1000 mg/kg/d and therefore the NOAEL for toxicity in this study was determined to be 1000 mg/kg/day for males and females.

The NOAEL for fertility parameters (functional or morphological) was 1000 mg/kg/d for both males and females in this study.