Registration Dossier

Administrative data

Description of key information

From the results of tis study it is concluded that the NOAEL for female Wistar rats after repeated administration of the registration substance for 90 consecutive days is 750 mg/kg body weight per day, the highest dose tested. At this dose level in female animals no significant clinical or systemic toxic findings occurred and no histopathological liver lesions were detected. Observed thyroid gland findings are considered to be of secondary nature. In males, due to the observed kidney lesions, no NOAEL could be established under the conditions of this study. However, the nature of kidney lesions was shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat-specific event and therefore without toxicological relevance to humans. Therefore, no signs of toxicological relevance for humans were demonstrated also in male animals up to a dose of 750 mg/kg body weight per day. The absence of relevant and/or significant systemic toxicity of the registration substance is supported by the results of a subacute oral toxicity study according to OECD TG 422 which revealed a NOAEL of 1000 mg/kg body weight per day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-22 to 2014-12-09
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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: 7-8 weeks old, females: 7-8 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 143 – 170 g (mean: 154.75 g, ± 20% = 123.80 – 185.70 g)
females: 119 – 143 g (mean: 128.87 g, ± 20% = 103.09 – 154.64 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. 1426 and 0902)
- 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. 240113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs
before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most
homogenous variation in body weight throughout the groups of males and females.

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
Preparation of the Test Item Formulations
The test item was weighed into a tarred plastic vial on a precision balance and the required volume of sesame oil was added.
Homogenization was achieved by vortexing for 2-3 minutes.
Before each dose administration homogeneity of the test item formulation was maintained by vortexing the prepared suspension thoroughly.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.

Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups and 1 control group:

Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 750 mg/kg body weight

Administration of Doses
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of 4 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 concentration of test item in dosing formulations, samples were retained from all groups once in the first week of
treatment, at beginning of month 2 and month 3, respectively, and at the end of the treatment period and stored between -15 and -35 °C.
Concentration was tested in all samples (16 samples in total).
Stability of the dosing formulations was tested once at the beginning of the treatment period. From all dose groups samples of dosing
formulations were frozen at 0 hours and 6 hours after preparation and stored between -15 and -35 °C. Stability was tested in samples of
dosing formulations of the low and high-dose groups only (4 samples in total).
In the first week of treatment, at the beginning of the second month of treatment and at the end of the treatment period, samples for the
testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high, medium and low-dose formulations and
stored between -15 and -35 °C. Homogeneity was tested in samples of dosing formulations of the low dose and high dose groups only
(18 samples in total).
Each sample was retained twice (sample A, sample B, each of at least 10 mL).
At the end of the treatment period all A samples of dosing formulations were handed over to the Principal Investigator and analysed at
BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 130407.
The samples B were retained at BSL and will be discarded after completion of the final study report.
Duration of treatment / exposure:
Period of 90 days
Frequency of treatment:
Once a day; 7 days per week
Remarks:
Doses / Concentrations:
100 mg/kg body weight; 300 mg/kg body weight; 750 mg/kg body weight;
Basis:
actual ingested
No. of animals per sex per dose:
100 animals (50 males and 50 females) were included in the study.
10 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender
of the control and of the high dose group were subjected to necropsy 28 days after the last administration (end of recovery period).
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days.
Ten 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 90 days after the last administration (end of recovery period).

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.
The animals in the control group were handled in an identical manner to the test group subjects and received sesame oil using the same volume
as used for the high dose group.
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 90 days. The recovery animals were observed for an additional
period of 90 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, public holidays and on 18 March 2013 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 last 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.
For female animals no. 51-55 (C), 61-65 (LD), 71-75 (MD) and 81-85 (HD) these tests were performed one week before the last week of exposure.

Examination of Fertility Parameters
Daily over a period of 8 days, the estrous cycle of all female animals, excluding the animals to be evaluated at the end of the recovery period,
were examined 4, 8 and 12 weeks after the first administration. In the recovery animals the estrous cycle was examined during the last week of the
recovery period. At necropsy (one day after the last administration) and at the end of the recovery period, one epididymis and one testis were
separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count were evaluated in
all male animals using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0C).

Haematology
Haematological parameters 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 deeply anaesthetized animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells (Luc).

Blood Coagulation
Coagulation parameters 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 deeply anaesthetized animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry were 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 of deeply anaesthetized 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
A urinalysis was performed with samples collected from all animals at necropsy.
The following parameters (specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL),
erythroctes (Ery), leukocytes (Leu)) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Additionally, urine colour / appearance were recorded.







Sacrifice and pathology:
Pathology
Gross necropsy
One day after the last administration (study day 91) all animals of the treatment period and 90 days after the last administration all animals
of the recovery period (study day 181) were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24139, expiry date: 06/2014 and xylazin,
Serumwerk, lot no. 00512, expiry date: 07/2014).
Blood was sampled before the animals were exsanguinated and subjected to detailed gross necropsy. During necropsy a careful examination
of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents were made.
Any organs showing abnormalities were preserved.

Organ Weight
The wet weight of the following organs (liver, uterus, kidneys, thymus, adrenal glands, thyroid/parathyroid glands, testes, spleen, epididymides,
brain, prostate gland, pituitary gland, seminal vesicles and coagulating glands after ligation, heart, ovaries) of all sacrificed animals was
recorded as soon as possible. Paired organs were weighed individually.
After weighing, one testis and one epididymis were separated and used for the evaluation of sperm parameters.
The following tissues (all gross lesions, lungs (inflated with fixative), brain (cerebrum, cerebellum and pons), aorta,
spinal cord (cervical, mid-thoracic and lumbar), uterus with cervix, pituitary, ovaries, thyroid/parathyroid glands, vagina, thymus, Testes (one),
oesophagus, epididymides (one), salivary glands, prostate gland (ventral and dorsal lobes), stomach, seminal vesicles, small and large intestines
(including Peyer´s patches), coagulating glands, liver, mammary glands (male and female), pancreas, urinary bladder, kidneys,
lymph nodes - (mesenteric and axillary lymph nodes), adrenal glands, peripheral nerve (sciatic nerve) with skeletal muscle, spleen,
bone with bone marrow (sternum), heart, skin, trachea, eyes) 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.

Histopathology
The organs listed (see above) were examined histopathologically 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. Additionally, liver, kidney, thyroid gland and lung were examined in all animals of the LD, MD and recovery groups.
Any gross lesion macroscopically identified have been examined microscopically.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional
hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Immunohistochemistry of α2µ-Globulin in Male Kidneys
Immunohistochemistry of α2µ-globulin was performed on kidney of all male animals sacrificed at the end of the treatment period of the study
(40 animals). The processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath
Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing).
Immunohistochemistry was performed with Rat α2µ-Globulin Antibody (Monoclonal Mouse IgG1 Clone #129736, R&D Systems). The image analysis
was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x20.
The positive area on the total area were measured in mm2 and calculated as % on the total area. The relative values of positive structures
(α2µ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum). Since descriptive statistics provide no
indication for higher levels of α2µ-globulin in kidney slides of high dose animals as compared with controls, no inferential statistics were performed.
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 (p<0.05 was considered as statistically
significant).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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):
Some parameters were affected but none of them is assumed to have any toxicological relevance
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):
Mainly liver and kidney by mechanisms not relevant for humans (α2μ-Globulin mediated) or no dose-dependency
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mainly α2μ-Globulin mediated effects without relevance for humans
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 and recovery period of this study.

Clinical Observations
Slight clinical signs observed immediately after administration, e.g. prone position, piloerection, salivation and/or moving of the nose through the bedding occurred transiently in all dose groups. These symptoms occurred only during the treatment period and are assumed to be related to local irritation of the test item formulation and are not considered a sign of systemic toxicity.

Ophthalmological investigations
There were no ophthalmoscopic findings in any of the animals of this study.

Body Weight Development
Mean body weight gain was mildly but statistically significantly attenuated in male – but not in female – animals at 750 mg/kg body weight. No significant difference was found in the body weight gain during the recovery period or in the low and mid dose groups of this study, when compared to the respective controls.

Food Consumption
The test item did not affect food consumption during the treatment period or recovery period 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.

Examination of Fertility Parameters
No effects on the reproductive system (e.g. organ weights, spermatogenesis, tubular stages of spermatogenic cycle, epididymal sperm motility, testicular sperm count) or histopathological changes of reproductive organs were observed.

Haematology and Blood Coagulation
At the end of the treatment period there were no biological relevant differences in haematological parameters of male and female animals
determined in this study. Besides, all remaining haematological parameters were within the normal range of variation and statistically
significant differences are not assumed to be biologically relevant.

Clinical Biochemistry
At the end of the treatment period no relevant differences were observed in hepatocellular markers (ALAT, ASAT). Statistical significant differences in the latter are not assumed to be biologically relevant. At the end of the recovery period no considerable difference in the above-mentioned serum markers for hepatotoxicity was observed between the high dose group and the control group. Statistically significant differences in ASAT and AP levels in female animals of the dose group are not assumed to be biologically relevant. Besides, all remaining haematological parameters were within the normal range of variation and statistically significant differences are not assumed to be biologically relevant.

Urinalysis
No effect of the test item on urinary parameters was found at the end of the treatment period and at the end of the recovery period.

Pathology
At the end of the treatment period kidneys were spotted in 3/10 male animals treated at 750 mg/kg body weight. None of the female animals of this
group were affected. Likewise, the thyroid/parathyroid gland was discolored in 3/10 male animals. Again, none of the female animals of this
group was affected. Single or occasional macroscopic additional findings are considered to be incidental findings.

Organ Weight
At the end of the treatment period the liver weight was statistically significant increased in animals of the high dose group. However, at the end of the recovery period no considerable difference in liver weight between high dose animals and controls was found. This finding is considered to be an adaptive response, most probably involving the induction of hepatic microsomal enzymes. Slightly increased absolute kidney weight was observed in male and to a lesser extend in female animals of the high dose. When related to body weight, kidney weight was also statistically significantly increased in male but not in female animals at a dose of 300 mg/kg body weight. Since the effect in female animals was minimal and no histopathological correlate was evident, this finding may be accidental and was considered not to be adverse. In high dose male animals, some kidneys were macroscopicall spotted and related histopathological kidney lesions were shown to be hyaline inclusions consiting of α2μ-Globulin, a male rat specific event without toxicological relevance to humans. Thyroid/parathyroid weight was slightly higher at the end of the treatment period in animals at 750 and 300 mg/kg body weight. The liver and thyroid gland changes recovered almost completely during the recovery period and are considered to be of secondary nature.

Histopathology
Terminal Sacrifice:
Histopathological findings clearly attributable to administration of the test item were seen in the kidney of males and in the liver and thyroid gland
of both sexes. Histopathological findings of questionable test item relationship were noted in the kidney of females and in the lung of both sexes.
The incidence and severity of hyaline droplets in the renal cortex was dose-relatedly higher in all three treated male groups, when compared with
controls. This lesion was associated with a minimal or mild corticotubular degeneration in most males, also in a dose-related manner. In the liver, a
centri- to panlobular hepatocellular hypertrophy was observed in a low number of males and females treated at 300 mg/kg/day
and in most animals treated at 750 mg/kg/day. The change was minimal in the mid dose group and minimal or mild in the high dose group and
confirmed the higher liver weights recorded. Other histopathological findings seen at terminal sacrifice were considered to be most likely incidental and/or to be within the range of expected changes for rats of this strain and age kept under similar experimental conditions.

Recovery Sacrifice
Kidney lesions had almost recovered after the treatment-free period. There was no longer any inter-group difference in the incidence or degree
of hyaline droplets, basophilic tubules or tubular casts, and dilated tubules were not observed at all. A minimal amount of golden-brown pigment
in corticotubular cells in males previously treated at 750 mg/kg/day was considered to be related to the process of recovery of previous renal
changes.

Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
From the results of tis study it is concluded that the NOAEL for female Wistar rats after repeated administration of the registration substance for 90 consecutive days is 750 mg/kg body weight per day, the highest dose tested. At this dose level in female animals no significant clinical or systemic toxic findings occurred and no histopathological liver lesions were detected. Observed thyroid gland findings are considered to be of secondary nature. In males, due to the observed kidney lesions, no NOAEL could be established under the conditions of this study. However, the nature of kidney lesions was shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat-specific event and therefore without toxicological relevance to humans. Therefore, no signs of toxicological relevance for humans were demonstrated also in male animals up to a dose of 750 mg/kg body weight per day.
Executive summary:

Oral administration of the registration substance via gavage to groups of 10 male and 10 female Wistar rats at doses of 100, 300 or 750 mg/kg body weight per day, for 90 consecutive days resulted in no mortality throughout the study period. A control group received the vehicle sesamoil. Slight clinical signs observed generally immediately after administration, e.g. prone position, piloerection, salivation and/or moving of the nose through the bedding, which occurred transiently in all dose groups are not considered to be of toxicological significance. No test item related effects on parameters of functional observation battery were found. Body temperature was not affected at the end of the treatment or recovery period. There were no ophthalmoscopic findings in any of the animals of this study. Body weight gain was mildly attenuated in male – but not in female – animals at 750 mg/kg at the end of the treatment period. The test item did not affect food consumption during the treatment or recovery period of this study. At the end of the treatment or recovery period of this study there were no toxicologically relevant alterations in haematological parameters in male and female animals. With regard to clinical chemistry, serum level of total bile acids was moderately decreased at 100, 300 and 750 mg/kg bw in male and female animals, and a slightly lower total bilirubin serum level was observed in male animals treated with 300 or 750 mg/kg body weight per day when compared to respective controls.The decrease of these hepatobilary parameters is assumed to be test item related but is generally not associated with hepatobiliary toxicity and no considerable alteration in these parameters was observed at the end of the recovery period. Moreover, no relevant differences were observed in hepatocellular markers (ALAT, ASAT). No effect of the test item on urinary parameters was found at the end of the treatment period and at the end of the recovery period. Concerning fertility parameters, no test item related effects on epididymidal sperm motility and testicular sperm count were found at the end of the treatment or recovery period in male animals and no alteration of cyclicity (length or sequence of stages) was observed in female animals at any dose group. At the end of the treatment period the liver weight was statistically significant increased in animals of the high dose group. However, at the end of the recovery period no considerable difference in liver weight between high dose animals and controls was found. This finding is considered to be an adaptive response, most probably involving the induction of hepatic microsomal enzymes. Slightly increased absolute kidney weight was observed in male and to a lesser extend in female animals of the high dose. When related to body weight, kidney weight was also statistically significantly increased in male but not in female animals at a dose of 300 mg/kg body weight. Since the effect in female animals was minimal and no histopathological correlate was evident, this finding may be accidental and was considered not to be adverse. In high dose male animals however, some kidneys were macroscopicall spotted and related histopathological kidney lesions were shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat specific event without toxicological relevance to humans. Thyroid / parathyroid weight was slightly higher at the end of the treatment period in animals at 750 and 300 mg/kg body weight. The liver and thyroid gland changes recovered almost completely during the recovery period and are considered to be of secondary nature. In the lung of both sexes a slight to moderate increase in alveolar macrophages was observed which was not considered to be adverse and intergroup differences were not present after the recovery period. In all dose groups, absolute and relative weight of prostate gland was moderately lower than in control animals. This was not associated with histopathologicaly findings. No higher prostate weight was observed at 750 mg/kg bw at the end of the recovery period and toxicological significance was not attributed to this finding.

Based on the findings of this study it is concluded that the NOAEL for female Wistar rats after repeated administration of the registration substance for 90 consecutive days is 750 mg/kg body weight per day. At this dose level in female animals no significant clinical or systemic toxic findings occurred and no histopathological liver lesions were detected. Observed thyroid gland findings are considered to be of secondary nature. In males, due to the observed kidney lesions, no NOAEL could be established under the conditions of this study. However, the nature of kidney lesions was shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat-specific event and therefore without toxicological relevance to humans. Therefore, no signs of toxicological relevance for humans were demonstrated also in male animals up to a dose of 750 mg/kg body weight per day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral administration of the registration substance via gavage to groups of 10 male and 10 female Wistar rats at doses of 100, 300 or 750 mg/kg body weight per day, for 90 consecutive days resulted in no mortality throughout the study period. A control group received the vehicle sesamoil.Slight clinical signs observed generally immediately after administration, e.g. prone position, piloerection, salivation and/or moving of the nose through the bedding, which occurred transiently in all dose groups are not considered to be of toxicological significance.No test item related effects on parameters of functional observation battery were found. Body temperature was not affected at the end of the treatment or recovery period. There were no ophthalmoscopic findings in any of the animals of this study. Body weight gain was mildly attenuated in male – but not in female – animals at 750 mg/kg at the end of the treatment period. The test item did not affect food consumption during the treatment or recovery period of this study. At the end of the treatment or recovery period of this study there were no toxicologically relevant alterations in haematological parameters in male and female animals. With regard to clinical chemistry, serum level of total bile acids was moderately decreased at 100, 300 and 750 mg/kg bw in male and female animals, and a slightly lower total bilirubin serum level was observed in male animals treated with 300 or 750 mg/kg body weight per day when compared to respective controls.The decrease of these hepatobilary parameters is assumed to be test item related but is generally not associated with hepatobiliary toxicity and no considerable alteration in these parameters was observed at the end of the recovery period. Moreover, no relevant differences were observed in hepatocellular markers (ALAT, ASAT). No effect of the test item on urinary parameters was found at the end of the treatment period and at the end of the recovery period. Concerning fertility parameters, no test item related effects on epididymidal sperm motility and testicular sperm count were found at the end of the treatment or recovery period in male animals and no alteration of cyclicity (length or sequence of stages) was observed in female animals at any dose group.At the end of the treatment period the liver weight was statistically significant increased in animals of the high dose group. However, at the end of the recovery period no considerable difference in liver weight between high dose animals and controls was found. This finding is considered to be an adaptive response, most probably involving the induction of hepatic microsomal enzymes. Slightly increased absolute kidney weight was observed in male and to a lesser extend in female animals of the high dose. When related to body weight, kidney weight was also statistically significantly increased in male but not in female animals at a dose of 300 mg/kg body weight. Since the effect in female animals was minimal and no histopathological correlate was evident, this finding may be accidental and was considered not to be adverse. In high dose male animals however, some kidneys were macroscopicall spotted and related histopathological kidney lesions were shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat specific event without toxicological relevance to humans. Thyroid / parathyroid weight was slightly higher at the end of the treatment period in animals at 750 and 300 mg/kg body weight. The liver and thyroid gland changes recovered almost completely during the recovery period and are considered to be of secondary nature. In the lung of both sexes a slight to moderate increase in alveolar macrophages was observed which was not considered to be adverse and intergroup differences were not present after the recovery period. In all dose groups, absolute and relative weight of prostate gland was moderately lower than in control animals. This was not associated with histopathologicaly findings. No higher prostate weight was observed at 750 mg/kg bw at the end of the recovery period and toxicological significance was not attributed to this finding.

Based on the findings of this study it is concluded that the NOAEL for female Wistar rats after repeated administration of the registration substance for 90 consecutive days is 750 mg/kg body weight per day. At this dose level in female animals no significant clinical or systemic toxic findings occurred and no histopathological liver lesions were detected. Observed thyroid gland findings are considered to be of secondary nature.In males, due to the observed kidney lesions, no NOAEL could be established under the conditions of this study. However, the nature of kidney lesions was shown to be hyaline inclusions consisting of α2μ-Globulin, a male rat-specific event and therefore without toxicological relevance to humans. Therefore, no signs of toxicological relevance for humans were demonstrated also in male animals up to a dose of 750 mg/kg body weight per day.

The absence of relevant and/or significant systemic toxic effects of the registration substance is also supported by a subacute oral toxicity test according to OECD TG 422. In this study the registration substance was administered to 12 Wistar rats/sex/dose level by oral gavage (4 mL/kg bw) at dose levels of 0 (vehicle PEG 400 only), 75, 300 or 1000 mg/kg body weight per day. Unmated recovery animals (5 rats/sex at control and high dose) were kept for further 14 days, without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. The study comprised also a reproductive/developmental toxicity screening, intended to provide information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on the development of the F1 offspring from conception to day 4 post partum. There were no test item-related effects in mortality, clinical signs, body weight, food consumption, ophthalmology, haematology, clinical chemistry, urinalysis, neurobehaviour, gross pathology, histopathology, reproductive or developmental endpoints. Transient liver and kidney weight changes noted at 300 and 1000 mg/kg bw/d were considered to represent adaptive responses. The NOAEL for repeated dose oral toxicity in parental animals was 1000 mg/kg body weight per day.

For risk assessment purposes the NOAEL of 750 mg/kg body weight per day from the subchronic 90 -day oral toxicity test is used.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study according to GLP

Justification for classification or non-classification

Based on the results of a subchronic 90 -day oral toxicity study in rats according to OECD TG 408 and supported by the absence of findings in a subacute repeated dose toxicity study according to OECD TG 422, the registration substance is not subject to classification and labelling requirements according to Directive 67/548/EEC (DSD) and Regulation 1272/2008/EC (CLP).