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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Feb 2017 to 17 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21st September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Directive 2001/59/EC, Official Journal of the European Communities, L225, 21.8.2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han IGS (Crl:WI(Han))
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. This rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6-7 weeks old at the start of the treatment period.
- Weight at study initiation: The body weights at initiation of treatment were within ± 20% of the mean weight for each sex, and ranged from 143- 171 g (mean 155 g) for males and from 113- 134 g (mean 123 g) for females.
- Housing: The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex. On the day of FOB testing and motor activity assessment, the animals were temporarily kept singly in macrolon cages. During urine collection, animals were kept singly in stainless-steel metabolism cages.
- Diet: From their arrival until the end of the study (unless precluded by the collection of concentrated urine in week 13, or the collection of blood from overnight fasted rats prior to scheduled necropsy), the rats received a powdered, cereal-based rodent diet (VRF1 (FG))from a commercial supplier (SDS Special Diets Services, Witham, England) batch 2771. The diets were provided ad libitum in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. During the study, the food in the cans was replaced by fresh portions from the freezer weekly and filled up as needed.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 Feb 2017 To: 16 (males) and 17 (females) May 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was used because this is a possible route of human exposure and the key route for assessing the toxicity of the substance as such.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials (one vial per group per day) in a refrigerator. The vehicle for dosing the controls wassimilarly be stored. All gavage liquids were continuously stirred on a magnetic stirrer during the dosing procedure, to ascertain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: 0, 6, 24 and 100 mg/mL, corresponding to dose levels of 0, 30, 120 and 500 mg/kg bw/ day respectively.
- Amount of vehicle (if gavage): 5 mL/kg bw/ day, the dose volumes were adjusted weekly to the latest recorded body weights for each individual rat.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the test substance in gavage liquid were conducted using a Gas Chromatography (GC) method, based on the method provided by the sponsor. The test substance was used as the reference standard for the preparation of calibration samples.

METHOD VALIDATION
Before analysis of study samples, the analytical method was validated by analyzing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996.
- Recovery: the mean recovery of the test substance from gavage liquid should be between 85% and 115% at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
- Specificity: signals should be corrected in case the signal obtained for blank samples was ≥ 5% of the signal obtained for low-dose samples..

ANALYSIS OF GAVAGE LIQUIDS
Preparation of validation samples:
Validation samples with nominal concentrations of 0, 6, 24 and 100 mg test substance per ml gavage liquid were prepared by addition of approximately 0, 12, 48 and 200 mg test substance to 2 ml gavage liquid (control, low-dose, mid-dose and high-dose, respectively).

Validation samples were analyzed in triplicate and study samples were analyzed in duplicate.

Homogeneity:
The homogeneity of the test substance was assessed in the batch of gavage liquids, prepared for the study. Three samples of each test gavage liquid, taken at different locations in the gavage liquid container, and 1 sample of the control gavage liquid were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the gavage liquid container). The test substance was considered to be homogeneously distributed in the gavage liquid if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.

Stability:
The stability of the test substance in gavage liquid was assessed in the batch of gavage
liquids, prepared for the study (4 hours animal room and 1 week at 2-10 °C). One sample of each test gavage liquid and one sample of the control gavage liquid were analyzed in duplicate.
For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in gavage liquid if p ≥ 0.01 and/or if the mean concentration after storage was within 90-110% of the mean concentration at t = 0.

Content:
The content of the test substance was determined in the batches of gavage liquids, prepared for the study five data spread evenly within the study period.
The content of the test substance in gavage liquid was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.

Duration of treatment / exposure:
13 consecutive weeks (7 days/week)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were intended to provide information on the sub-chronic oral toxicity of the test substance and to establish a no-observed-adverse-effect level (NOAEL).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment (including post-dosing signs) were recorded.

DETAILED CLINICAL OBSERVATIONS AND NEUROBEHAVIOURAL EXAMINATION:
In addition to the above daily general clinical observations, detailed clinical examinations (in an arena outside the home cage) were performed on all rats prior to the first exposure and then once weekly throughout the study. Behavioral endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study (in week 12 or 13 for females and in week 13 for males). Motor activity tests data recorded on DVD were removed from the study dossier after submission of the final report.

BODY WEIGHT:
The body weight of each animal was recorded once during the acclimatization period (day -1), at initiation of treatment (day 0), and once per week thereafter. The animals were weighed after fasting prior to scheduled necropsy in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage by weighing the feeders. The consumption was measured over weekly periods of for all animals in the cage. The results were expressed in g per animal per day.

WATER CONSUMPTION
Water consumption was measured per cage, by weighing the drinking bottles daily, during 5- day periods in weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of treatment (on day -5) in all rats, and in the last week of the treatment period (on day 83 and 85 for males and females, respectively) in all rats of the control group and the high-dose group. Because no treatment related ocular changes were observed in the high-dose group, eye examination was not extended to the animals of the intermediate-dose groups at the end of the study. Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulfate.

HAEMATOLOGY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). EDTA or citrate (for prothrombin time) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out: hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells (Lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time and thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were discarded after analysis. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, (fasting) glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate (PO4).

URINALYSIS:
In week 13 (day 84-85 and 85-86 for males and females, respectively), all rats were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were individually kept in metabolism cages and urine was collected. Urine samples were discarded after analysis. The following determinations were carried out in individual samples: volume and specific gravity (to investigate the concentrating ability of the kidneys), appearance, pH, glucose, microscopy of the urinary sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, worm eggs, sperm cells), occult blood, ketones, protein, bilirubin and urobilinogen.
Sacrifice and pathology:
GROSS PATHOLOGY:
Early in week 14, after overnight fasting (water was freely available), the animals were killed on two successive working days (day 91 and 92 for males and females, respectively) in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anesthesia and then examined grossly for pathological changes.
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated on the basis of the terminal body weight of the animals: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (with coagulating glands), spleen, testes, thymus, uterus.

HISTOPATHOLOGY:
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin.
Histopathological examination (by light microscopy) was performed on all tissues and organs listed below, of all animals of the control group (1) and the high-dose group (4) with the exception that gross lesions, liver and spleen from all animals (groups 1-4) were examined. The kidneys were examined in all male rats of all dose groups. Additionally, renal α2- microglobulin was assessed by immunohistochemistry. For that purpose, sections of the kidneys of all male animals of all dose groups were processed for immunohistochemical staining of α2-microglobulin using an α2-microglobulin antibody (Thermofisher PA5-47788).

The following organs and tissues were examined: adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum, cerebellum), cecum, colon, duodenum, epididymides, esophagus, eyes, GALT (gut associated lymphoid tissue, including Peyer's patches), heart, ileum, jejunum, kidneys, liver, lungs, mammary gland (females), mesenteric lymph nodes, nerve-peripheral (sciatic), ovaries, oviducts (=fallopian tubes), pancreas, parathyroid, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles+ coagulating glands, skeletal muscle (thigh), skin (flank), spinal cord (retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar)), spleen, sternum with bone marrow, stomach (non glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix), vagina, all gross lesions.
Statistics:
Please refer to 'other information on materials and methods incl. tables'

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted mainly in the high-dose group and occasionally also in the lower dose groups. This finding is ascribed to the dosing of the rats by oral gavage. Post dosing signs comprised salivation just prior to and/or after dosing and, at a few occasions, sliding with the ventral parts of the head and neck over the cage bottom after dosing. These findings were noted mainly in the high-dose group and occurred only incidentally in the other groups. There were no other treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were statistically significantly decreased in males of the low- and mid-dose group at several stages. The differences with the controls were only slight (between 3-8%) and there was no dose-response relationship. Body weights were statistically significantly increased in females of the high-dose group and (at two occasions) in the mid-dose group. The differences with the controls were very slight (less than 5%). There were no marked differences in body weight gain between test groups and controls. Occasionally, statistically significant differences in body weight gain were noted, but these were not consistent. In conclusion, there were no relevant changes in growth parameters.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was increased in the high-dose group in both sexes. The differences with the controls were statistically significant at most of the weekly stages. Incidentally, a statistically significant difference with controls also occurred in the low- or middose group, but these findings were not consistent and the overall food intake was not affected in these groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake was increased in the high-dose group in both sexes. In the first week of the study, the differences with the controls were only slight (overall 8%) and occurred in highdose males only. At the subsequent stages, these differences aggravated (overall ≥ 41% for males and ≥ 24% for females) and were often statistically significant. In the other groups water intake was not affected; an incidental statistically significant decrease in water intake in low-dose males on day 5 of the study was considered a chance finding.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any treatment-related changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group, statistically significant decreases were noted in red blood cell count, hemoglobin concentration and packed cell volume in males, and in hemoglobin concentration in females. Thrombocytes were increased in males of the high-dose group. Prothrombin time was reduced in females of the high-dose group. An incidental increase in MCHC was noted in males of the low-dose group.
Total white blood cell count was statistically significantly increased in males of the high-dose group. The absolute counts of lymphocytes, neutrophils and basophils were also increased but there were no relevant changes in the percentage distribution.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol and phospholipids were statistically significantly increased in females of the highdose group. The values obtained in this group were outside the range of historical control data.
The following changes were statistically significant but were not considered to be of toxicological significance:
- Ca concentration was increased in the high-dose group in both sexes. Ca concentration was also increased in low-dose males but there was no dose response relationship. The values in the various groups were within the range of historical control data and therefore the fluctuations in calcium level were not considered to be adverse.
- ASAT activity was decreased in females of the high-dose group. However, an increase rather than a decrease in this variable may represent a toxic effect .
- Creatinine concentration was decreased in females of the high-dose group.
- An incidental decrease in Albumin/globulin ratio was noted in mid-dose males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The renal concentration test showed a statistically significantly increased urinary volume in males of the high-dose group. The specific gravity was not significantly affected. This finding was probably related to the higher water intake in the high-dose group. Semi-quantitative (dipstick) urinary measurements showed a slight though statistically significant decrease in urinary pH in males of the mid- and high-dose group. Protein and ketones were decreased in males of the high-dose group. These findings are not considered to be of toxicological significance. Urinary appearance (color and clarity) is presented in individual animals only. There were no clear differences in urinary appearance among the groups. Microscopic examination of the urinary sediment showed a statistically significant increase in epithelial cells, amorphous material and casts in males of the high-dose group. The casts were noted in six males of this group and also in two males of the mid-dose group. In females of the high-dose group, urinary crystals were increased, but there was considerable variation in the severity of this finding in individual rats in the various groups. Urinary crystals are considered of limited, if any, toxicological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the substance in rats.
Immunological findings:
no effects observed
Description (incidence and severity):
In general, the results of this study did not indicate any immunologica effects (in or decrease of white blood cells)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences with the controls were noted:
- The relative weight of the liver showed a dose-related increase in males and females of the mid- and high-dose groups. The differences with the controls were 12-15% in the mid-dose group and more than 50% in the high-dose group. The absolute liver weights were increased in high-dose males and in mid- and high-dose females.
- The relative weight of the kidneys was increased in males of the mid- and high-dose groups (13% and 36%, respectively) and in females of the high-dose group (13%). The absolute kidney weights were increased in high-dose males and in mid- and high-dose females.
- The relative weight of the spleen was increased (37%) in males of the high-dose group. The absolute weight of the spleen was also increased in males of this group.
- The relative weight of the heart was increased (11%) in males of the high-dose group.

The following changes were statistically significant but were not considered to be of toxicological significance:
- The absolute weight of the heart was decreased in males of the mid-dose group. This finding was not reflected in significant changes in the relative weight of this organ and is therefore considered a chance finding.
- The relative weight of the testes was increased in mid-dose males. This finding is ascribed to the lower terminal body weights in this group. During growth retardation, rats tend to maintain their (absolute) weight of the testes, and there is a well-known inverse correlation between terminal body weight and relative testes weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy no treatment related macroscopic changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation revealed treatment-related microscopic changes in the kidneys, the liver and the spleen.

- The microscopic changes in the kidneys comprised intra- and extracellular accumulation of hyaline droplets in tubules of the outer cortex of the kidneys of 10/10 high-dose males, in all cases accompanied by an increased number of basophilic tubules. In seven of these males the kidneys also showed dilated tubules with eosinophilic casts consisting of granular debris. The characteristics of the findings in the kidneys, along with the observation that they occurred in males only, were highly suggestive for accumulation of alpha 2 urinary microglobulins (alpha 2u). To further specify these changes the kidneys were immunohistochemically stained with a polyclonal antibody against alpha 2u. Evaluation of the immunohistochemically stained kidneys revealed that the structures identified as hyaline droplets and granular casts in the HE stained slides showed a positive reaction (in Table 15 indicated as ‘ihc (immunohistochemically) positive for alpha-2u’). Microscopy of the kidneys of the low- and mid-dose males revealed alpha 2u-positive hyaline droplet accumulation in 9/1 low-dose males and in 10/10 mid-dose males. The severity of these findings was generally highest in the high-dose group and lower in the low- and mid-dose groups.
- The microscopic changes in the liver were characterized by centrilobular hepatocellular hypertrophy (enlarged cells as well as enlarged nuclei) and hepatocellular vacuolation. The incidence of hepatocellular hypertrophy was 3/10, 6/10 and 9/10 in the low-, mid- and highdose males, respectively and 8/10 and 9/10 in the mid- and high-dose females, respectively. The incidence of macro-vesicular hepatocellular vacuolation was increased in 10/10 mid-dose and 10/10 high-dose females.
- The microscopic changes in the spleen were characterized by increase of extramedullary erythropoiesis in 8/10 high-dose males, in 3/10 mid-dose females and in 5/10 high-dose females.

The other organs and tissues did not reveal treatment-related histopathological changes. The histopathological changes observed were about equally distributed between the high-dose group and the controls or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Analysis of the dosing dilutions

- The substance was homogeneously distributed in the carrier at each test concentration.

- The substance was stable in the carrier under the experimental conditions (storage in the animal room for 4 hours or in the refrigerator (2-10°C for 7 days).

- The content of the substance in the test dilutions was close to intended in all five batches analysed, except for the low-dose level prepared on 13 March 2016 (the difference with intended was -16%). This finding was, however, attributed to the relatively high intercept of the calibration curve obtained during this analytical run.

- On the basis of the above it is concluded that the rats received the intended concentrations.

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of the substance, for a period of 13 weeks to Wister Han IGS (Crl:WI(Han)) rats revealed a NOAEL of 120 mg/kg bw/day based on toxicological significant effects on liver and spleen in the high dose group.
Executive summary:

The key study is a sub-chronic (13-week) repeated dose toxicity study performed according to OECD TG 408 and GLP principles, rated Klimisch 1. Wistar Han IGS rats (Crl:WI(Han)) were administered daily by oral gavage at dose levels of 30, 120 and 500 mg/kg bw/day. A control group treated with vehicle (corn oil) was included. In each dose group 10 male and 10 female animals were included. Analytical verification of dose and stability of the test in vehicle showed that the rats received the intended concentrations. All parameters are measured in accordance with the current guideline.

Clinical signs and body weights: There was no mortality. Salivation was noted just prior to or after dosing, mainly in the high dose group. There were no other treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Ophthalmoscopic examination did not reveal any treatment related ocular changes. There were no relevant changes in growth parameters. Food and water intake were increased in the high-dose group in both sexes.

Haematology: Haematology parameters were studied in all rats at necropsy. In the high-dose group haemoglobin concentration was decreased in both sexes, and red blood cell count and packed cell volume were decreased in males at maximum with 12%. Thrombocytes were increased in males, and prothrombin time was reduced in females of this group. Total white blood cell counts were increased in males of the high-dose group.

Clinical chemistry was conducted in all rats at necropsy. An increase cholesterol and phospholipids concentrations in females of the high-dose group was seen.

Urinalysis, conducted in all rats in week 13 of the study, did not reveal relevant changes in renal concentrating ability or in semi-quantitative (dipstick) urinary measurements. Microscopic examination of the urinary sediment showed increases in epithelial cells, amorphous material and casts in males of the high-dose group. Casts were also noted in two males of the mid-dose group.

Organs:

Spleen: the relative weight of the spleen (37%) was increased in males of the high-dose group. Macroscopic examination at necropsy revealed no treatment-related changes. The changes in the spleen were characterized by increased extramedullary erythropoiesis in 8/10 high-dose males, in 3/10 mid-dose females and in 5/10 high-dose females. Extramedullary erythropoiesis in the spleen may be regarded as a physiological mechanism to meet an increased demand for the production of red blood cells. However, a reason for the increased demand could not be established; the bone marrow did not show abnormalities indicating that the normal production of red blood cells was impaired. Further, no signs of an increased red blood cell turnover were found (such as haemorrhages, elevated reticulocytes or increased accumulation of iron pigment in the spleen). Taking into account that the extramedullary erythropoiesis noted in a few females in the mid-dose group was not accompanied by functional disturbances in red blood cell system, this finding in the mid-dose group is not considered adverse.

Liver: The relative weight of the liver was increased in males and females of the mid- and high dose groups (12-15% in the mid-dose group and 50% in the high-dose group). The microscopic changes in the liver were characterized by centrilobular hepatocellular hypertrophy (enlarged cells as well as enlarged nuclei) and hepatocellular vacuolation. The incidence of centrilobular hepatocellular hypertrophy was 3/10, 6/10 and 9/10 in the low-, mid- and high-dose males, respectively; and 8/10 and 9/10 in the mid- and high-dose females, respectively. In addition, 10/10 mid-dose and 10/10 high-dose females showed a minimal increased incidence of macro-vesicular hepatocellular vacuolation. This minimal vacuolation was seen in all groups in the males with a lower incidence. The incidence of hepatocellular hypertrophy in the liver showed a clear dose-effect relationship in males of all dose groups and in females of the mid- and high dose group. The elevated blood lipids, increase in relative liver weight in combination with hepatocellular hypertrophy. These effects may be considered a reaction to an increased metabolic demand. Liver weight increase and hepatocellular hypertrophy in the absence of clinical pathology or histologic alterations indicative of overt hepatocellular damage may be considered an adaptive non-adverse effect ESTP Expert Workshops (2012, 2016). This may be concluded for the high dose too but in view of the high relative liver weight increase (and accompanying effects) the mid-dose is considered non-adverse for liver effects.

Kidney: The relative weight of the kidneys was increased in males of the mid- and high-dose groups (13% and 36%, respectively) and in females of the high-dose group (13%). Microscopy revealed alpha 2urinary microglobulins (accumulation of hyaline droplets in tubules of the outer cortex of the kidneys accompanied by an increased number of basophilic tubules, and/or dilated tubules with eosinophilic casts consisting of granular debris) and identified by immunohistochemical staining.

Other effects: There were no neurotoxicity and immunological toxic findings in this study. There were also no neoplastic findings in this study.

Conclusion

In conclusion, a NOAEL of 120 mg/kg bw/day could be derived based on the effects in the spleen and liver which were accompanied by changes in haematology or clinical biochemistry in the high dose group only.