Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.02.2012 - 19.03.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST 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 allocation of the animals to the experimental groups: males:162 - 179 g, (mean: 170.6 g, ± 20% = 136.48 – 204.72 g),
females:126 - 151 g, (mean: 137.9 g, ± 20% = 110.32 – 165.48 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.

ENVIRONMENTAL 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. 1145 and 1114)
- 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 individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

IN-LIFE DATES: From: 07.02.2012 To: 09.03.2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Diprom, Batch No.:10952-1, Expiry Date: 09/2013
Details on oral exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 28 days. 5 animals per gender and
group were subjected to necropsy one day after the last administration (end of treatment period).
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the nominal concentration of test item in dosing formulations, samples were retained from all groups once weekly
during the treatment period and stored between -15°C and – 35°C (16 samples in total).
In the first week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared
high and low dose formulations and stored between -15°C and – 35°C (6 samples in total).
Dose formulations were sampled for stability analysis. In first week of the study, samples of dosing formulations from LD and HD groups
were frozen at (0 hr) and 6 hours after preparation and stored at -15 and -35 °C (4 samples in total).
The chemical analyses were performed at BSL BIOSERVICE.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days per week for a period of 28 days
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg bw, 300 mg/kg bw, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
40 animals (20 males and 20 females) were included in the study (5 male and 5 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but no death
or severe suffering. Thereafter, a descending sequence of dose levels was selected with
a view to demonstrate any dosage related response and NOAEL.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time
each day and considering the peak period of anticipated effects after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups,
on the first day of administration and weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination, using an ophthalmoscope was made on all animals
before the first administration and in the last week of the treatment period.

HAEMATOLOGY: Yes
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the 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)

CLINICAL CHEMISTRY: Yes
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the 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)

URINALYSIS: Yes
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded. The following parameters were measured using qualitative indicators:
specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu).

NEUROBEHAVIOURAL EXAMINATION: Yes
Once before the first exposure and once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.

OTHER:
Blood Coagulation
Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the 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)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
One day after the last administration (study day 29) all surviving animals of the treatment period were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 23623, expiry date: 07/2013 and xylazin, Serumwerk, lot no. 00311, expiry date: 03/2013) 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.

HISTOPATHOLOGY: Yes
The organs of all animals listed in Table 7 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. All gross lesions identified in any animal were examined. Histological processing of tissues to microscope slides was performed at the GLP certified contract laboratory Propath UK Ltd. (test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites. The principal histopathological investigator of the test site provided the histopathology results to the study director by e-mail to be integrated in the report. The principal investigator sent a pathology phase report to the study director on completion of the study.
Other examinations:
The wet weight of the following organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately. 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
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
of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software
(p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Predominant clinical signs observed in the HD group were salivation and moving the bedding. 5/5 male HD animals exhibited a blue skin – this occurred most probably due to the blue color of the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
Predominant clinical signs observed in the HD group were salivation and moving the bedding. 5/5 male HD animals exhibited a blue skin – this occurred most probably due to the blue color of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight tendency towards a reduced body weight gain in the mean value of male HD animals between Day 15 and 22 occurred mostly because of a low value of animal No. 19.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Several parameters were dose-dependently but just slightly affected
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Some organs were slightly affected
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A blue discoloration of several organs and tissues like skin, kidney, testes, epididymides, and liver was recognized in MD and HD animals
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological findings were restricted to liver, stomach. kidney and testis (non adverse, please refer to the details given below).
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Observations
All male and female animals showed test item-related clinical signs in the HD groups. Predominant clinical signs observed in the HD group were
salivation and moving the bedding. 5/5 male HD animals exhibited a blue skin – this occurred most probably due to the blue color of the test item.
The clinical findings in the C, LD, and MD groups were assumed to be incidentally or are assumed to occur due to mild stress because of the
application procedure per se.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In males the mean body weight increased with the progress of the study in the C, LD, MD, and HD groups. In females LD, MD, and HD groups,
the mean body weight increased, too. In female C animals a slight loss in body weight was measured between days 22-28. This is attributed to
the application procedure per se. A slight tendency towards a reduced body weight gain in the mean value of male HD animals between Day 15
and 22 occurred mostly because of a low value of animal No. 19. However, since this animal has a normal body weight gain during the other
measured periods, it is assumed to be incidental.
Throughout the treatment period, body weights were within the normal range of variation for this strain.

Food Consumption
No considerable effect of FAT 40853/A TE on food consumption was found in any of the groups of male and female animals.

Haematology and Blood Coagulation
No biological relevant change was measured for the values of haematology of the male treatment groups at the end of the treatment period.
Statistical significant changes are assumed to be not biological relevant for the male animals.
Blood coagulation values were unchanged for male MD and HD animals when compared to the C animals. The mean value of aPTT of the male LD
animals was slightly increased when compared to the C animals – however, this value was still in the range to what could be expected regarding
our historical C data. Furthermore, this increase mainly belongs to two individual LD animals (No. 7 and 8). The other values of 3/5 male LD
animals were in the range of the C values. Since only 2/5 male LD animals and no MD or HD animals were affected, these increased ara assumed
to be incidental.
Female mean values of PLT were slightly but dose-dependently increased in LD, MD, and HD groups. However, all values are still in the range to
what could be expected when considering our historical C data. Hence, this increase is not of toxicological relevance. All other values of
haematology were not clearly changed in the treatment groups when compared to the C animals.
In case of coagulation of the female animals, just 1 value could be considered for PT in C animals, 2 values for PT in LD animals, 3 values for PT in
MD animals and 2 values for PT in HD animals. No value could be measured for aPTT in C animals, 2 values for aPTT in LD animals, 3 animals for
aPTT in MD animals and 1 animal for aPTT in HD animals. The missing values were either not measurable because of already clotted samples or
because of the sample did not clot during the measurement. However, since this happened also at the C group, is probable that this occurred
because of a technical problem during the blood withdrawal. Hence it is assumed that this finding is not test item related. All remaining values
within all groups are in the range of our historical C data and are not different among all groups. Hence, no test item related influence can be
considered for blood coagulation.

Clinical Biochemistry
At the end of the treatment period male values of ASAT were slightly but dose dependently increased in the male treatment groups. However, this increase was just slight and mainly attributed to 2 individual animals (No. 14 and No. 19) in the MD and HD group which exhibited high values. Hence, these increases are not considered to be of toxicological relevance.
Male urea values were significantly decreased (p<0.05) in HD animals. However, the mean value of the HD group was still in the range to what can be expected regarding our historical C data. Hence, this increase is not considered to be of toxicological relevance. Values of TBA are decreased in treatment groups when compared to C group. Furthermore, values of cholesterol are significantly increased (p<0.01) in male HD animals indicating an affected cholesterol elimination from the blood due to the low amount of bile acids. However, all parameters were within the normal range of variation for this strain. Note, that HD values of male animals of TBIL could not be measured due to a technical problem. The exact reason remains unclear. However, a problem due to the blue colouration of the serum because of the blue test item seems probable.
Female values of ASAT were slightly but dose dependently decreased in treatment groups. However, all values were still in the range of our historical C data. Values of AP of female animals were dose dependently decreased, too. Again, these values were still in the range to what could be expected regarding our historical C data. Values of TBIL were dose dependently decreased in LD and MD group. The values were still in the range of our historical C data. However, the TBIL amount of the HD animals could not be measured – most probably because of the blue colouration of the serum due to the blue colour of the test item. Values of TBA are not clearly affected in treatment groups since a high variability was observed in treatment groups.
Values of glucose are decreased in treatment groups which was not dose dependent but statistically significant in LD and MD groups. The values were still in the range of our historical C data. Besides, all parameters of clinical chemistry of female treatment groups were within the normal range of variation for this strain. For female animals no clear toxicological relevance can be attributed.


Urinalysis
2/5 male animals of the MD as well as 3/5 male animals of the HD group exhibited a low but measurable amount of ketone in the urine.
This appearance of ketone was most probably due to the starvation of the animals and hence is considered to be not test item related. 1/5 male
animals (No. 18) exhibited a very high value of erythrocytes in the urine. This was assumed to be incidental since no other animal exhibited the
same finding. 1/5 female HD animals (No. 37) exhibited a high specific gravity for the urine. Since no other animal was found exhibitibg
this finding it is assumed to be incidental. Erythrocytes in the urine of female animals were variable throughout the groups.
Hence, this is assumed to be incidental. All other urine parameters exhibited no finding in the treatment groups.

Pathology
One specific gross pathological finding was recorded for the male and female animals which was considered to be treatment-related – a blue
discoloration of several organs and tissues like skin, kidney, testes, epididymides, and liver. Mostly affected were animals in the HD group but
also MD animals. This occurred due to the blue color of the test item. An individual male animal in LD and one in the MD group exhibited yellow
spots on the right epididymides which were assumed to be incidental. In female animals a little extra tissues was found below the liver in an
HD animal which is assumed to be incidental. Further incidental findings were a discolored dark thymus as well as a discolored red axilliary
lymph node.

Organ Weight
In male animals a decrease in absolute thymus weights was found for MD and HD animals. These results were confirmed by the male relative
thymus weights (to brain weight and to body weight). Although no clear statistically significance as well as no clear dose dependency was observed, this change is assumed to be test item related as well as toxicologically relevant. Thymus weights are a sensitive parameter of generally stress due
to treatment with a test substance.
Absolute prostate weights (including seminal vesicles and coagulating gland) were decreased in male HD animals. Again this was confirmed by the
relative prostate weights of the HD animals (to brain weight and to body weight). Even no statistically significance is given this finding could be of
toxicological relevance.
The absolute weights of male thyroid/parathyroid gland were decreased in LD, MD, and HD animals. This was confirmed by relative weights of
thyroid/parathyroid gland (to body weight and brain weight). However, since this decrease was relatively slight and since no dose dependency
was observed this finding is assumed to be incidental. Group means of the absolute weights of pituitary gland of male animals were strongly
decreased in treatment groups. This was confirmed by relative organ weights (to brain weight and to body weight). This mainly depends on a
very high organ weight of the pituitary gland of C animal No. 1. Furthermore animal No. 4 of the C group also exhibited a relatively high weight
of the pituitary gland. Both high weights are assumed to be incidental and because of this, no toxicological relevant impact on the weight of this
organ in treatment groups can be considered. No other organ weight of the male animals was considerably affected in this study – neither
absolute nor relative organ weights. Absolute liver weights of female animals are slightly increased in HD animals which could be partly c
onfirmed by relative organ weights (to brain and to body weight). However, this increase is very slight and has as an individual finding no toxicological relevance. Weights of ovaries of female animals were increased in HD animals of HD group (absolute weights and relative weights to brain
weight and body weight). This could be of toxicological relevance. Weights of uteri of female animals of treatment groups are variable.
Whereas an increase in weight can be found for LD and MD animals, a decreased weight could be observed for HD animals – for absolute weights
as well as relative weights (to brain and body weight). Although this variance is remarkable it is not assumed to be of toxicological relevance.
Absolute as well as relative weights (to brain and body weight) of thyroid weights of female animals were dose-dependently decreased in
treatment groups. Due to the dose dependency as well the strong decrease in HD group (43 % for absolute weights) this finding is assumed
to be of toxicological relevance.
Absolute and relative weights (to brain weight and to body weight) of the pituitary gland were variable among the treatment groups.
Since no consistent weight change among the treatment groups could be observed, this finding is assumed to be not of toxicological relevance.

Histopathology
Histopathological findings were restricted to the liver, stomach, kidney and testis. In the liver, minimal diffuse bile duct hyperplasia arising in the portal region of the liver was observed in the mid- and high-dose group in a dose-related manner. Neither degenerative nor necrotic changes were observed in the bile ducts nor cholestasis. Based on severity of the abnormality and the fact that ductular proliferation often represents a reparative effect on the bile duct system which is reversible after withdrawal of the agent (Hailey et al., 2013), minimal bile duct hyperplasia is not considered as predictive of potential hepatotoxicity. In the stomach, histopathological findings were seen in all dose groups and were roughly dose-related. In the nonglandular mucosa, findings comprised combinations of diffuse hyperkeratosis, diffuse epithelial hyperplasia and epithelial hyperplasia of the limiting ridge, in all dose groups and in a roughly dose-related manner. They were mostly minor in degree and considered indicative of local irritation by the test item formulation. In the glandular mucosa, changes were characterized by diffuse mucous cell hyperplasia, decreased number of chief cells, multifocal mucosal single cell death, multifocal mucosal eosinophilic droplets and inflammatory submucosal edema. These lesions were considered to be most likely indicative of a local irritant effect of the test item formulation. Minor amounts of grey pigment were seen within the corticotubular
epithelium of the kidney at 1000 mg/kg/day and in a single female at 300 mg/kg/day, as well as in interstitial macrophages in the testis
at 1000 mg/kg/day. These changes were considered to be related to test item deposition and to be without toxicological significance.
In the mesenteric lymph node, minimal or mild sinus histiocytosis was observed in a proportion of animals treated at 300 or 1000 mg/kg/day and in one single female treated at 100 mg/kg/day. Treatment relationship of this finding remained questionable.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study week 1, 2, 3, and 4 for all dose groups. The mean recoveries observed
in LD, MD and HD groups were 94.3%, 97.5% and 96.3% of the nominal concentration, respectively. Stability of formulation samples was determined
in study week 1 for LD and HD groups. The mean recoveries observed after 6 hours storage at room temperature for LD group was 102.2% and
for HD group 99.8% of the concentration without storage. Homogeneity of formulation samples was determined in study week 1 for LD and HD
groups. The mean recoveries observed (during week 1) for LD group was 93.2% and and for HD group was 94.4% of the nominal value.
The coefficients of variation of the different sampling locations (top, middle, bottom) in LD group was 1.7% and in HD group also 1.7%.

Effect levels

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 relevant signs of systemic toxicity observed in test animals

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No relevant signs of systemic toxicity were observed in the animals treated at 100 mg/kg/day, 300 mg/kg/day and 1000 mg/kg/day. Thus the dose level of 1000 mg/kg/day can be considered as NOAEL for systemic toxicity in the conducted study.

The gastric findings were most probably caused by a local irritant effect of the test item formulation when administered repeatedly by oral gavage and are therefore not considered to be relevant for risk evaluation in humans.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of FAT 40853/A TE via oral administration to rats over a period of 28 days. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but receivedaqua ad injectionem, the vehicle used in this study. The 4 groups comprised of 5 male and 5 female rats. During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured twice weekly.

At the end of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations of these organs were extended to animals of the medium and low dose groups if treatment-related changes were observed in high dose group. The following doses were evaluated:Control: 0 mg/kg body weight, Low Dose: 100 mg/kg body weight,Medium Dose: 300  mg/kg body weight, High Dose: 1000  mg/kg body weight. The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionemand administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.

No mortality occurred in the control or any of the dose groups during the treatment period of this study. All male and female animals showed test item-related clinical signs in the HD groups. Predominant clinical signs observed in the HD group were salivation and moving the bedding. 5/5 male HD animals exhibited a blue skin – this occurred most probably due to the blue color of the test item. There were no ophthalmoscopic findings in any of the animals of this study. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. In female C animals a slight loss in body weight was measured between days 22-28. This is attributed to the application procedureper se. A slight tendency towards a reduced body weight gain in the mean value of male HD animals between Day 15 and 22 occurred mostly because of a low value of animal No. 19. However, since this animal has a normal body weight gain during the other measured periods, it is assumed to be incidental. No considerable effect of FAT 40853/A TE on food consumption was found in any of the groups of male and female animals. No biological relevant change was measured for the values of haematology of the male treatment groups at the end of the treatment period. Blood coagulation values were unchanged for male MD and HD animals when compared to the C animals. The mean value of a PTT of the male LD animals was slightly increased when compared to the C animals – however, this value was still in the range to what could be expected regarding our historical C data. Female mean values of PLT were slightly but dose-dependently increased in LD, MD, and HD groups. However, all values are still in the range to what could be expected when considering our historical C data. Hence, no test item related influence can be considered for blood coagulation of female animals. At the end of the treatment period male values of ASAT were slightly but dose dependently increased in male treatment groups. Male urea values were slightly but significantly decreased (p<0.05) in HD animals. Values of TBA are decreased in treatment groups when compared to C group. Furthermore, values of cholesterol are significantly increased (p<0.01) in male HD animals indicating an affected cholesterol elimination from the blood due to the low amount of bile acids. Female values of ASAT were slightly but dose dependently decreased in treatment groups. Values of AP of female animals were dose dependently decreased, too. Values of TBIL were slightly but dose dependently decreased in LD and MD group. Values of glucose are decreased in treatment groups which was not dose dependent but statistically significant in LD and MD groups. No treatment related influence was seen for the values of urine in male or female treatment groups.

One specific gross pathological finding was recorded for the male and female animals which was considered to be treatment-related – a blue discoloration of several organs and tissues like skin, kidney, testes, epididymides, and liver. Mostly affected were animals in the HD group but also MD animals. In male animals a decrease in absolute thymus weights was found for MD and HD animals. These results were confirmed by the male relative thymus weights (to brain weight and to body weight). Absolute prostate weights (including seminal vesicles and coagulating gland) were decreased in male HD animals. Again this was confirmed by the relative prostate weights of the HD animals (to brain weight and to body weight). The absolute weights of male thyroid/parathyroid gland were slightly decreased in LD, MD, and HD animals.

Absolute liver weights of female animals are slightly increased in HD animals which could be partly confirmed by relative organ weights (to brain and to body weight). Weights of ovaries of female animals were increased in HD animals of HD group (absolute weights and relative weights to brain weight and body weight). Absolute as well as relative weights (to brain and body weight) of thyroid weights of female animals were dose-dependently decreased in treatment groups.

Further, roughly dose-related effects in the stomach indicative for local irritations due to test item deposition and substance application were determined in both genders among the test groups starting in the low-dose group. As food consumption and body weight development appeared normal in affected animals, systemic toxicity based on the effects observed in the stomach is excluded. Minor amounts of grey pigment were seen within the corticotubular epithelium of the kidney in the high-dose animals of both sexes and in a single mid-dose female without accompanying effects on the organ. Moreover, interstitial macrophages were present in the testes of high-dose males associated with test item deposition. In the mesenteric lymph node, minimal or mild sinus histiocytosis was observed in 1/5 males of mid- and high-dose groups and in 1/5 low-, 2/5 mid- and 3/5-high-dose females which might be related to test item deposition. Moreover, minimal diffuse bile duct hyperplasia arising in the portal region of the liver was observed in the mid- and high-dose group in a dose-related manner. Neither degenerative nor necrotic changes were observed in the bile ducts nor cholestasis. Based on severity of the abnormality and the fact that ductular proliferation often represents a reparative effect on the bile duct system which is reversible after withdrawal of the agent (Hailey et al., 2013), minimal bile duct hyperplasia is not considered as predictive of potential hepatotoxicity. Moreover, animals of the mid- and high-dose group did not show any evidence of functional impact on the liver which exacerbates the identification of discernible toxic effects. In detail, decreased levels of ALP, glucose, TBA and increased ASAT do not provide sufficient evidence for liver toxicity; especially as liver toxicity is generally associated with elevated TB, ALP and TBA levels. Therefore, alterations observed in clinical parameters are not considered as reliable indicators for functional impact on the liver. Taking further into account that minimal bile duct hyperplasia is considered as non-adverse in rats with little to no concern to humans (Hailey et al., 2013), minimal bile duct hyperplasia induced by the test substance is considered non-adverse, especially as 1) bile duct hyperplasia is known to occur in rats as a common spontaneous change which does not functionally impact the liver, 2) minimal ductular proliferation is often reparative, not carcinogenic and reversible (Hailey et al., 2014).

On the basis of this 28-Day Repeated Dose Oral Toxicity study with FAT 40853/A TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day, no biologically relevant signs of systemic toxicity were observed in the animals treated at 100 mg/kg/day, 300 mg/kg/day and 1000 mg/kg/day. Thus, the dose level of 1000 mg/kg/day can be considered as NOAEL for systemic toxicity in the conducted study. The gastric findings were most probably caused by a local irritant effect of the test item formulation when administered repeatedly by oral gavage and are therefore not considered to be relevant for risk evaluation in humans.