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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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System:

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: 6-7 weeks old, females: 6-7 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 163 - 203 g (mean: 184.97 g, ± 20% = 147.97 – 221.96 g)
females: 129 - 161 g (mean: 142.33 g, ± 20% = 113.87 – 170.80 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.

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. 1039)
- 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 fiber bedding (lot no. 190612).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test item formulation and vehicle were administered at a single dose to the animals by oral gavage.
The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose)
and 1 control group (C). The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.

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 animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the
same volume as used for the high dose group.

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 and -35 °C (Total 16 samples).
Dose formulations were sampled for stability analysis. In first week of the study, samples of dosing formulations from all group were frozen at
(0 hr) and 6 hours after preparation and stored at -15 and -35 °C (Total 4 samples).
In the first and fourth 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 and -35 °C (Total 12 samples).
Sample quantity for all samples was 5 mL in a 15 mL falcon tube.
At the end of the treatment period, all samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific
Laboratories GmbH and stored between -15 and -35 °C until analysis.
Formulation analysis were performed in accordance with GLP and the procedures followed for the determination of test item concentration in
the dosing formulations and control formulation were described in a separate study phase plan issued by the Principal Investigator which was
amended to study plan. The results were reported as separate phase report in the annex of the final report.
Duration of treatment / exposure:
The animals of the main and recovery groups were treated with the test item or vehicle on 7 days per week for a period of 28 days.
Five animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period).
Five animals per gender of the C and of the HD group were subjected to necropsy 14 days after the last administration (end of recovery period).
Frequency of treatment:
The animals were treated once daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg bw, 300 mg/kg bw and 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 main study (5 male and 5 female animals per group).
The main study included one control (C) and three dose groups (Low Dose = LD, Medium Dose = MD, High Dose = HD).
In addition, 20 animals (5 male and 5 female animals per group) were included in the control and high dose groups to be observed for
a period of 14 days following the last administration (Control Recovery = CR and High Dose Recovery = HDR).
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 administration.
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. 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
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 of tests. These tests were conducted
in all animals.
Haematology
Haematological parameters (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))
were examined at the end of the treatment and recovery period 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.

Blood Coagulation
Coagulation parameters (prothrombin time (PT), activated partial thromboplastin time (aPTT)) were examined at the end of the treatment
and recovery period 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.

Clinical Biochemistry
Parameters of clinical biochemistry (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)) were examined at the end of the treatment and recovery period 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.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.
The following parameters (specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood,
leukocytes) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Sacrifice and pathology:
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, medistar Arzneimittel, lot no: 00312,
expiry date: 06/2014 and xylazin, Serumwerk, lot no. 00312, expiry date: 05/2014 and lot no. 00512, expiry date: 07/2014) 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 organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands, testes, spleen, epididymides,
brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries,heart) 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.

The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), eye, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles with
coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus, lymphnodes
(mesentric and axillary), thyroid glands, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen, sternum with bone marrow,
lung and trachea, pituitary gland, mammary glands, oesophagus, skin) from all animals were preserved in 10% neutral buffered formalin
except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were
transferred to 10% neutral buffered formalin.All animals found dead and intercurrently euthanised for animal welfare reasons
were also subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology
The afore-listed organs (see above) 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.
The histopathological examinations were not extended to animals of all other dosage groups and recovery animals as no treatment-related
changes were observed in the high dose group.
A detailed qualitative examination of the testes from all males (main study) were made taking into account the tubular stages of the
spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. The examination was conducted
in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinuclear or apoptotic
germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
A detailed qualitative examination of the testes from recovery males was not performed as no treatment related changes were observed
in high dose group main study males.
Any gross lesion macroscopically identified was examined microscopically.
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 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 .

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. Statistical comparisons of data acquired during the recovery period
were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software or E-Workbook 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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
One female animal from the HD group (46) and one female from HDR (60) group died during the 28-day treatment period and one female
from HD (48) group was euthanized on study day18 due to animal welfare reasons. No mortalities were observed in other dose groups.
Based on prominent histopathological lesions in the respiratory system, death of the 3 females was considered to be due to gavage accident
and local irritant effect of the test item formulation.

Clinical Observations
No test item related clinical signs were observed in any male and female animal during the entire study period. However, few predominant
spontaneous clinical signs observed occasionally in male and female HD animals were abnormal breathing, moving the bedding, diarrhoea,
salivation, piloerection, nasal discharge and reduced spontaneous activity. Findings like alopecia and echar on various body parts was
either seen only occasionally in treatment groups or also occurred in the control group male and females.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
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 both males and females, no statistically or biologically significant effect was observed on body weight and body weight gain in treatment
groups during the entire study period. All group mean values for body weight and body weight gain were comparable with respective controls
and were within the normal range of variation for this strain. The mean body weight increased with the progress of the study in all groups.

Food Consumption
In correlation to the body weight and body weight gain, the food consumption (g/day) in both males and females was comparable to
controls in all groups. However, from week 2 in the treatment group females, the food consumption was marginally decreased during
treatment period when compared with controls.
As decrease was marginal and no effect on body weight was observed during entire study period, this marginal decrease in food consumption
was considered to be non- adverse.

Haematology and Blood Coagulation
In males, no statistically significant difference in any of haematological parameter was observed at the end of treatment period and recovery
period and all haematological parameters values were within the normal range of variation when compared with controls.
In females, at the end of the treatment period, statistically significant increase in reticulocytes in HD group was observed when compared with
control. This increase in female reticulocytes was considered to be non adverse as the individual values were within the normal range of
variation for this strain.
No statistically significant difference in any of other female haematological parameter was observed at the end of treatment period and
recovery period and all haematological parameters values were within the normal range of variation.
Blood coagulation was not affected by the test substance in both males and females.

Clinical Biochemistry
In males, statistical analysis of clinical biochemistry data at the end of treatment period revealed significant increase in potassium in
LD and MD group when compared with controls.
In females, at the end of the treatment period, statistically significant increase in urea in HD group was observed when compared with controls.
In males, due to lack of dose dependency, this increase in potassium in LD and MD was considered to be biologically irrelevant and of no
toxicological significance.
In females, urea individual values from HD group were still in the normal range of variation for this strain and therefore this statistically
significant increase was not assumed to be toxicologically relevant.

No statistically significant difference in any of other male and female clinical biochemistry parameter was observed at the end of treatment
period and recovery period and values for all clinical biochemistry parameters were within the normal range of variation.

Urinalysis
High protein and erythrocytes levels were found in the urine of few male and female animals of all groups including control.
Besides, all urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control
group were observed.

Pathology
Few specific gross pathological changes were recorded in female animals at terminal or recovery sacrifice and were not considered to be
treatment-related. Findings were assumed to be common background findings in this strain.
Predominant macroscopic findings observed in male animals were yellow spot on right epididymides (1/5 in control and MD group).
In females, animals with fluid distension in uterus (2/5 in LD, MD and 3/5 in HD), fluid distension in lung (1/5 in HD), gas filled digestive
tract (1/5 in HD) and dark discolouration of liver, spleen, lung and thymus (1/5 in HDR group) were observed.

Organ Weight
In males, at the end of recovery period, statistically significant decrease in relative (to brain weight) epididymides weight was observed in
HDR group when compared to respective control group.
In females, at the end of treatment period and recovery period, no statistically significant difference in absolute and relative (to brain and
body weight) organ weight data from treatment group was observed when compared with controls.
The decrease in relative (to brain weight) epididymides weights in HDR males sacrificed at the end of recovery period was considered to
be toxicologically irrelevant as no such effect was observed in the males sacrificed at the end of treatment period.

Histopathology
Three females treated at 1000 mg/kg/day did not survive the treatment period. Based on prominent histopathological lesions in the
respiratory system, their death was considered to be due to gavage accident and local irritant effect of the test item formulation.
No test item-related changes of toxicological relevance were noted in rats sacrificed at terminal sacrifice. One male treated at 1000 mg/kg/day
showed mild epithelial alteration of the trachea, which was considered to be most probably indicative of a preceding (possibly partial)
inadvertent intra-tracheal instillation of the test item formulation. It was therefore not considered to be toxicologically relevant.
Animals sacrificed at the end of the recovery period were not evaluated histologically as no treatment related changes were observed
in high dose group.
As a conclusion, under the conditions of this study, and based on the fact that changes of the respiratory system were not considered
relevant for risk assessment in humans, the NOAEL (No Observed Adverse Effect Level) for pathology was considered to be 1000 mg/kg/day.

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 101%, 98% and 98% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study weeks 1 and 4 for LD and HD dose groups. The mean recoveries observed for
LD group were 101 and 97%, for HD group 96 and 101% of the nominal value. The coefficients of variation of the different sampling locations
(top, middle, bottom) in LD group were 1.9 and 7.5% and in HD group 1.5 and 1.4%.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item related effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the repeated oral administration of the test item to male and female Wistar rats at doses of
100, 300 and 1000 mg/kg body weight for 28 days was not associated with major signs of toxicity. However, one female animal from the HD
group and one female from HDR group died during the 28-day treatment period and one female from HD group was euthanized on study day 18
due to animal welfare reasons. Histopathologically, their deaths were considered to be due to gavage accident and local irritant effect
of the test item formulation.
No test item related effect on clinical signs, body weight, food consumption, urine parameters, hematology, clinical biochemistry parameters,
organ weight, gross pathological findings and histopathology was observed up to 1000 mg/kg body weight/day.
Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the substance is considered to be 1000 mg/kg
body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test item 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(sterile water), the vehicle used in this study.

The 4 groups comprised of 5 male and 5 female Wistar rats.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from

toxic effects, animals in the recovery group were observed for a period of 14 days following the last administration.

Body weight and food consumption were measured weekly.

At the conclusion 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. These examinations were not extended to animals of all

other dosage groups and recovery groups as no treatment-related changes were observed in the high dose group. Gross lesions macroscopically identified were

examined microscopically in all animals.

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 suspended inaqua ad injectionem (sterile water) and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.


 

Summary Results

Mortality

One female animal from the HD group and one female from HDR group died during the 28-day treatment period and one female from HD group was euthanized due to animal welfare reasons. No mortalities were observed in other dose groups.Based on prominent histopathological lesions in the respiratory system, death of the 3 females was considered to be due to gavage accident and local irritant effect of the test item formulation.

Clinical Signs

No test item related clinical signs were observed in anymale and femaleanimal during the entire study period.However, few predominant spontaneous clinical signs observed occasionally in male and female HD animals were abnormal breathing, moving the bedding, diarrhoea, salivation, piloerection, nasal discharge and reduced spontaneous activity.Findings like alopecia and eschar on various body parts was either seen only occasionally in treatment groups or also occurred in the control group male and females.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

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 wereno ophthalmoscopic findings in any of the animals of this study.

Body Weight Development

In both males and females, no statistically or biologically significant effect was observed on body weight and body weight gain in treatment groups during the entire study period. All group mean values for body weight and body weight gain were comparable with respective controls and were within the normal range of variation for this strain. The mean body weight increased with the progress of the study in all groups.

Food Consumption

In correlation to the body weight and body weight gain, the food consumption (g/day) in both males and females was comparable to controls in all groups. However, from week 2 in the treatment group females, the food consumption was marginally decreased during treatment period when compared with controls.

Haematology and Blood Coagulation

In males, no statistically significant difference in any of male haematological parameter was observed at the end of treatment period and recovery period and all haematological parameters values were within the normal range of variation when compared with controls.

No statistically significant difference of toxicological relevance in female haematological parameter was observed at the end of treatment period and recovery period and all haematological parameters values were within the normal range of variation.

Blood coagulation was not affected by test item in both males and females.

Clinical Biochemistry

No statistically significant difference of toxicological relevance in male and female clinical biochemistry parameter was observed at the end of treatment period and recovery period and values for all clinical biochemistry parameters were within the normal range of variation.

Urinalysis

High protein and erythrocytes levels were found in the urine of few male and female animals of all groups including control. Besides, all urinary parameters were in the normal range of variation and no conspicuous differences between dose group and control group were observed.

Pathology

Few specific gross pathological changes were recorded in female animalsat terminal or recovery sacrificeand were not considered to be treatment-related. Findings were assumed to be common background findings in this strain.

Predominant macroscopic findings observed in few male animals were yellow spot on right epididymides. In females, animals with fluid distension in uterus, fluid distension in lung, gas filled digestive tract and dark discolouration of liver, spleen, lung and thymus was observed.

Organ Weight

In males, at the end of recovery period,no statistically significant difference of toxicological relevance was observedwhen compared to respective control group.

In females, at the end of treatment period and recovery period, no statistically significant difference in absolute and relative (to brain and body weight) organ weight data from treatment group was observed when compared with controls.

Histopathology

No test item-related changes of toxicological relevance were noted in rats sacrificed at terminal sacrifice. One male treated at 1000 mg/kg/day showed mild epithelial alteration of the trachea, which was considered to be most probably indicative of a preceding (possibly partial) inadvertent intra-tracheal instillation of the test item formulation. It was therefore not considered to be toxicologically relevant.

As a conclusion, under the conditions of this study, and based on the fact that changes of the respiratory system were not considered relevant for risk assessment in humans, the NOAEL (No Observed Adverse Effect Level) for pathology was considered to be 1000 mg/kg/day.

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 101%, 98% and 98% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study weeks 1 and 4 for LD and HD dose groups. The mean recoveries observed for LD group were 101 and 97%, for HD group 96 and 101% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) in LD group were 1.9 and 7.5% and in HD group 1.5 and 1.4%.

Conclusion Under the conditions of the present study, the repeated oral administration of the test substance to male and female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight for 28 days was not associated with major signs of toxicity. However,one female animal from the HD group and one female from HDR group died during the 28-day treatment period and one female from HD group was euthanized on study day18 due to animal welfare reasons. Histopathologically, their deaths were considered to be due to gavage accident and local irritant effect of the test item formulation. No test item related effect on clinical signs, body weight, food consumption, urine parameters, hematology, clinical biochemistry parameters, organ weight, gross pathological findings and histopathology was observed up to 1000 mg/kgbody weight/day. Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the substance is considered to be 1000 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.