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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed in accordance with validated guidelines and in GLP compilance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(4-bromophenyl)-2-methylpropionic acid methyl ester
EC Number:
450-630-9
EC Name:
2-(4-bromophenyl)-2-methylpropionic acid methyl ester
Cas Number:
154825-97-5
Molecular formula:
C11H13BrO2
IUPAC Name:
methyl 2-(4-bromophenyl)-2-methylpropanoate
Test material form:
other: liquid
Details on test material:
The test item was liquid with a degree of purity >99%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were derived from a controlled full barrier maintained breeding system (spf). No specific findings during the adaptation period; females nulliparous, non-pregnant. 5 female and 5 male rats were evaluated at each dose level. In total 20 females, 20 males.

Body weight at the commencement of the study:
Females: 135 - 163 g (mean: 148 g ± 20% = 118-177 g)
Males: 148- 175 g (mean: 163 g ± 20% = 130-195 g)
Age at the commencement of the study: 7-9 weeks
The animals were barrier maintained (semi-barrier) in an air conditioned room
Temperature: 22 ± 3°C
Rel. humidity: 55 ± 10%
Artificial light sequence being 12 hours light, 12 hours dark
Air change: 10 x/hour
Feeding ad libitum, Altromin 1324 maintenance diet for rats and mice, totally-pathogen-free (TPF)
Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
The animals were kept in Macrolon cages on Altromin saw fiber bedding
Adequate acclimatization period
Certificates of food, water and bedding are filed

The animals were randomly assigned to the dose/control groups according to a table of randomized numbers for coding and caged individually.
Prior to the first application a detailed clinical observation was made outside the cages with all animals.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
castor oil
Details on oral exposure:
The test item was dissolved in Corn Oil. The vehicle was chosen due to its non-toxic characteristics.
The single dosages (vigorously shaken) were prepared on every application day in the following ratios:

Low Dose (150 mg/kg BW): The test item was dissolved in Corn Oil at a ratio of 0.6 g to 20 mL.
Medium Dose (500 mg/kg BW): The test item was suspended in Corn Oil at a ratio of 2.0 g to 20 mL.
High Dose (750 mg/kg BW): The test item was suspended in Corn Oil at a ratio of 3.0 g to 20 mL.

The homogeneity of the preparations was visually checked. During the application procedure no obvious separation was detected.
Analysis of Stability and Homogeneity was performed.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
The animals were dosed with the test item on 7 days per week for a period of 28 days by gavage, using a stomach tube. In total 28 doses were administered per animal and group.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 mg/Kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/Kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/Kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 500 mg/kg bw/day
Male: 5 animals at 750 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 500 mg/kg bw/day
Female: 5 animals at 750 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
Preparation of the test item
The test item was dissolved in Com Oil. The vehicle was chosen due to its non-toxic characteristics. The single dosages (vigorously shaken) were prepared on every application day in the following ratios:
Low Dose (150 mg/kg BW): The test item was dissolved in Com Oil at a ratio of 0.6 g to 20 mL.
Medium Dose (500 mg/kg BW): The test item was suspended in Com Oil at a ratio of 2.0 g to 20 mL.
High Dose (750 mg/kg BW): The test item was suspended in Com Oil at a ratio of 3.0 g to 20 mL.
The homogeneity of the preparations was visually checked. During the application procedure no obvious separation was detected.

Analysis of Stability and Homogeneity of the test item in the vehicle was performed.

Administration of doses
The animals were dosed with the test item on 7 days per week for a period of 28 days by gavage, using a stomach tube. In total 28 doses were administered per animal and group.
Volume of application: The different doses were applied according to bodyweight at a volume of 5 ml/kg BW.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle in the identical volume.

The healt status of any animals was controlled. No specific findings during the adaptation period; females nulliparous, non-pregnant.

5 female and 5 male rats were evaluated at each dose level. In total 20 females, 20 males.

The animals were randomly assigned to the dose/control groups according to a table of randomized numbers for coding and caged individually.
Prior to the first application a detailed clinical observation was made outside the cages with all animals.

Examinations

Observations and examinations performed and frequency:
Body Weight Development
The animals were weighed prior to first application (day 0) and once a week thereafter (day 7, 14, 21, 27).
The total and weekly mean weight gain for each group was calculated.
The weights determined on the day of sacrifice were used for the calculation of the relative organ weights.

Food Consumption
Food consumption was calculated weekly (day 7, 14, 21, 27) with determination of food supply and residual.
The total mean and daily food consumption was detetmined for each group.

Clinical Observation
The observation period was 28 days. General clinical observations were made at least once a day, preferably at the same time each day. The health
condition of the animals was recorded. At least twice daily, all animals were observed for morbidity and mortality.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea,
changes in the skin and fur, eyes and mucous membranes (salivation, discharge ), piloerection and pupil size.
Once before the first exposure and in the final week, detailed clinical observations were made in all animals with specific emphasis on locomotion and
behaviour. These observations were made outside the home cage at the same time.

Behaviour/Functional Observation
Once before the first exposure and in the fourth exposure week sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive
stimuli, and motor activity assessment) was conducted. The functional observational battery was oriented according to the method described by Moser et
al., 1991 (Moser V.C., K.L. McDaniel, P.M. Phillips, Rat Strain and Stock Comparisons Using a Functional Observational Battery, Toxicol. Appl. Pharmacol.
108,267-283, 1991).
The examination began with a brief home cage observation, describing the pasture, palpebral closure, and the presence of convulsions or tremors. The
rat's reactivity to removal from the cage and handling were then rated and the observer noted presence of salivation, lacrimation, and piloerection.
Next the rat was placed on an open field (on a table surrounded by a 10 cm cardboard rim) for 3 min. during which time the arousal level (alertness) and
gait characteristics were recorded. The number of rears, supported (using the cardboard rim as support) and unsupported (unassisted), were counted
separately.
In addition, fecal boluses and pools of urine were counted. The presence of convulsions and tremors was again noted.
Reactions to the approach of a pencil, touch on the rump, and tail pinch using forceps were noted. The pupillary response was tested with the aid of a
red light using a penlight stimulus.
Flexion reflex, equilibrium, grasping reflex (together with grip strength), righting reflexes and auditory startle were tested. Finally the rectal body
temperature was taken.

Blood Sampling
The withdrawal of blood was performed by puncture of the abdominal aorta of the anaesthesized animals after overnight fasting. This was part of the
procedure of killing the animals on the day of necropsy.
The blood was collected into small tubes containing EDTA for haematology, citrat for clotting tests and plain tubes for clinical biochemistry.

Urine Sampling
The urine was collected into plain tubes by puncture of the urine bladder as a part of the necropsy.

Haematology and Clinical Biochemistry
Determined parameters:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Total leucocyte count (WBC)
Platelet count
APTT/PTT
Differential leucocyte count
AST (GOT) aspartate aminotransferase
ALT (GPT) alanine aminotransferase
Alkaline Phosphatase (AP)
Cholesterol (Chol)
Total Protein (TP)
Glucose (GLU)
Urea
Creatinine (CREA)
Albumin (ALB)
Na
K
Boehringer Combur Urin sticks (10 parameters)

Pathology
All animals in the study were subjected to a full, detailed gross necropsy which included careful examination of the extemal surface of the body, all
orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
The wet weight was taken from the following tissues immediately after preparation:
liver
kidneys
adrenals
testes
epididymides
thymus
spleen
brain
heart

Calculation of the Relative Organ Weight
The relative organ weight was calculated in relation to the bodyweights determined on the day of sacrifice and was expressed in %.

Organ Preparation
The following tissues were preserved in buffered (sodium dihydrogen orthophosphate/disodium hydrogen orthophosphate) 10% formalin solution from
all animals of all groups. It is the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination:
all gross lesions
brain (representative regions including cerebrum, cerebellum and pons)
spinal card
liver
kidneys
adrenals
stomach
small and large intestines (including Peyer' s patches, Lymphnodes acc. to application)
thymus
thyroid
spleen
lung and trachea
heart
gonads
accessory sex organs (e.g. uterus, prostate, vesicula seminalis)
urinary bladder
lymph nodes (Lymphocentrum mandibulare; Lnn axillares)
peripheral nerve
bone marrow

Histopathology
Full histopathology was carried out on the preserved organs and tissues of all animals in the Control, Medium Dose and High Dose groups.
Autolysis was noted for the tissues of high dose animals, due to a technical problem of tissue fixation (insufficient formalin concentration) and was documented. As the evaluation of high dose group tissues was limited by this autolysis, a full evaluation of tissues of the medium dose group was performed.
Additionally Liver, Thymus and Thyroids were evaluated from all animals in the Low Dose groups.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) was carried out, followed by Student's t-test to reveal any differences between control and test groups. For parameters indicating no compound related alterations and/or showing a high degree of variation (both in control and test groups) no statistical evaluation was carried out. Therefore, biological relevance was discussed.
In the evaluation of laboratory parameters all values within a range of the mean - value ± the two fold standard deviation (x ± 2s) are considered to be normal values within a normal population.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two male animals of the High Dose were found dead after the 12 th application, without showing clinical symptoms before All further animals (control- and test groups) survived throughout the test period and were sacrificed on day 28.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two male animals of the High Dose were found dead after the 12 th application, without showing clinical symptoms before All further animals (control- and test groups) survived throughout the test period and were sacrificed on day 28.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Increased weight gain was observed for the female HD group, compared to the control, reaching statistical significance (on the 95% confidence level).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Increased food consumption was calculated far the female HD group, compared to the control, reaching statistical significance (on the 95% confidence level).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant differences in haemoglobin, haematocrit and Red Blood Count (RBC) are found (on the 95% confidence level) for both HD groups. Significant differences in White Blood Count (WBC) and platelet are found for female and male HD groups respectively.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No relevant differences between test and control groups were found. No dose dependency and toxicological relevance was found.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Only in male animals at high dose hepatomegaly and 16 yellowish content in the small intestine and animal revealed a bloody lung.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Based on the results of the pathological evaluation, the NOEL (NoObserved- Effect-Level) was considered to be 150 mg/kg/day
Details on results:
Two male animals of the High Dose were found dead after the 12th application, without showing clinical symptoms before. The cause of death could not be determined for these two animals.
All further animals (control-and test groups) survived throughout the test period and were sacrificed on day 28. Beside the values in the female HD group, the mean weight gain in all groups was almost as high, as expected according to the standard growth curve for this strain. Individually diminished weight gains are due to the daily treatment associated with mechanical manipulation and therefore with increased stress for the animals. Increased weight gain was observed for the female HD group, compared to the control, reaching statistical significance (on the 95% confidence level).
In addition increased food consumption was calculated for the female HD group, compared to the control. All further animals showed normal food intake and no significant reduction in food consumption was found.
Although no clear significant toxicological relevance can be concluded, compound relation cannot be exluded.
No differences were observed conceming functional and behaviomal examination prior to application and during the last week of dosing, respectively.
No abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalization.
No convulsions, tremors, stereotypy or bizarre behaviour were observed. Animals were used to handling during week 4 as it could be expected. Responses to reflex testing were normal. Upon necropsy hepatomegaly in one HD animal was recorded. This finding is supported by organ weight findings as well as by the
histopathological assessment and is clearly compound related. The yellowish content in the small intestine noted for the two decendent male High Dose animals was considered to be not treatment related.

Upon histopathology the following is to be discussed:
Both decedents showed follicle center necrosis and/or lymphocytolysis of lymphoid organs, as well as epididymal oligospermia (in one male together with reduced height of testicular germinal epithelium). Based on the histopathological evaluation, their cause of death could not be established, and findings in lymphoid and reproductive organs were considered to represent most likely secondary unspecific changes due to general toxicity and treatment-related stress.
Renal cortical tubulonephrosis and hypocellularity of the bone marrow were observed in males and females treated at the HD of 750 mg/kg/day, sacrificed at study termination. Although interpretation was limited by the occurrence of tissue autolysis, these findings are considered to be toxicologically relevant.
Focal cystic degeneration (spongiosis hepatis) of the liver was seen in 3/5 females treated at 750 mg/kg/day. Although this change may occur in the untreated aging rat liver, it has also been described as an induced change after treatment with certain chemical compounds, amongst which hepatocarcinogens. In the present study, it is therefore considered toxicologically relevant.

The minimal to mild centrilobular to panlobular hepatocellular hypertrophy of the liver in both sexes treated at 500 (MD) or 750 (HD) mg/kg/day confirmed some macroscopic liver observations and statistically significantly higher liver weights measured in these groups. This hepatic change was considered to be an adaptive metabolic response of the liver to treatment with FEXO-03, including induction of hepatic microsomal enzymes.
Moreover, in two male rats treated at 500 mg/kg/day, there was a minimal follicular cell hypertrophy of the thyroid gland, interpreted to be the consequence of a
hormonal feedback mechanism caused by an increased hepatic clearance of thyroid hormones.
Upon the assessment of organ weights the most dominant findings were the increased values for the organ weights of the livers. Additionally findings in spleen, kidneys and thymus are to be discussed.

The mean absolute and relative liver weight in the MD and HD groups of both sexes was clearly increased compared to the weights in the corresponding Control groups, reaching statistical significance. This finding clearly corresponds to other findings (histopathology as discussed above, possibly liver enzymes) and is considered to be a clearly compound related effect with toxicological relevance. A clear dose dependency can be observed.
The mean absolute and relative thymus weight in the male MD and HD groups was clearly decreased compared to the weight in the corresponding Control group, reaching statistical significance for both values in the MD group and the absolute values in the HD group. This finding corresponds to histopathology results (minimal atrophy) and is considered to be a possible compound related effect with toxicological relevance, although of low incidence and severity at histopathological evaluation. There is a dose-dependent increase of absolute and relative kidney weight reaching statistical significance for the absolute and relative values of female HD and MD and the relative values for female LD and male HD. This finding corresponds to histopathology for the HD groups and is considered to be a compound related effect for these groups with toxicological relevance.
For the decreased spleen weight observed in the male HD group no clear toxicological relevance can be concluded.

Beside these findings no toxicological relevant results in relative and absolute organ weights for both sexes and any of the groups concerning the other organs have been found.
In the assessment of the haematology-values (Hct, Hb, RBC, WBC, Platelet) no severe deviations between the control- and dosed groups were found.
Overall no clear compound-relation and dose dependency was detectable. For the female HD group increase of the WBC values was observed, compared to the control. Although these values are all within the expected range, dose dependency may be considered. Toxicological relevance for further single deviations could not be concluded and are considered to be incidental.
For both HD groups (female and male) increase of the PTT values were found, reaching statistical significance for the male HD group, possibly indicative of a slight finding in the clotting system.
For the differential blood count no compound-relation and no results of toxicological relevance were found.

With the exception of the Glucose values, the determination of clinical chemical parameters revealed that most mean and many individual values were within the expected range. Statistically significant differences were found between some dose groups and the corresponding Control groups both in male and female animals. As none of the mean and individual values per se showed marked pathological deviations, significant differences in general represent variation within a normal range and are of no toxicological relevance. Some individual deviations may also be explained by technical reasons (e.g. problems while blood sampling) or individual, incidental disturbance. Some deviations are discussed below:

All mean and individual AST values are within the expected range (20-110 U/1). The calculated significance for the male LD and fernale HD groups does not correspond with toxicological significance, as all values are found to be within the range and deviation is only marginal.
All mean and individual ALT values are within the expected range (13-57 U/1). But, although all values are found to be within the range, treatment relation cannot be excluded, as increase is observed for the values of both HD groups and findings are confirmed by histopathological evaluation.

With the exception of single low values in (also in both control groups), all mean and all individual AP values are within the expected range (48-274 U/1
for females, 48- 323 U/1 for males).
Although all values are found to be within the range, dose dependency is clearly observed (compared to the corresponding control). Together with the ALT findings, liver organ weight findings and histopathological evaluation the result is indicative of a hepatopathic disturbance.
Most mean and most individual GLU values of all dosed groups are at the upper limit, or slightly above the expected range (5-10 mmol/L). Clear dose dependency is observed, esp. for the female MD and HD groups. As considered to be no marked finding, severe toxicological relevance cannot be concluded.
In the determination of the Urine-values no relevant differences between test- and control groups were found. No dose dependency and toxicological relevance was found.


In the surviving animals, clearly treatment-related findings were observed in the kidney, bone marrow, liver and thyroid gland.
Moderate renal cortical tubulonephrosis, as well as minimal or mild bone marrow hypocellularity were seen in both sexes treated at 750 mg/kg/day. In the liver, a focal cystic degeneration was noted in a proportion offemales treated at 750 mg/kg/day.
A minimal or mild centrilobular to panlobular hepatocellular hypertrophy of the liver was seen in the majority of males and females treated at 500 or 750 mg/kg/day, confirming some macroscopic liver observations and statistically significantly higher liver weights measured in these groups. This was considered to be an adaptive response to treatment, including induction of hepatic microsomal enzymes. In two males treated at 500 mg/kg/day, there was follicular cell hypertrophy of the thyroid gland, interpreted to be the consequence of a hormonal feedback mechanism caused by an increased hepatic clearance of thyroid hormones.
Minimal thymic atrophy or indistinct cortico-medullary border was observed in occasional animals of all treatment groups, but was not considered to be clearly treatment-related.

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The low dose of 150 mg/Kg bw is the no observed adverse effect dose level (NOAEL) of FEXO-03 dissolved in Corn Oil after a total of 28 applications by gavage over a period of 28 days.
Clear dose dependency was observed in mean liver, thyroid thyrnus and kidney findings, with a marked decrease in the severity of the findings evident from the high dose to the low dose.
A proper evaluation includes consideration of any correlation between haematological data, organ weight findings and histopathology. Incidental findings in LD animals of both sexes were of no clear toxicological significance.
The autolysis noted for tissues of high dose animals did not influence the evaluation of the study and deterrnination of a NOAEL.