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Diss Factsheets

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:
5 Feb to 11 Jun 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethoxydimethylsilane
EC Number:
214-189-4
EC Name:
Dimethoxydimethylsilane
Cas Number:
1112-39-6
Molecular formula:
C4H12O2Si
IUPAC Name:
dimethoxydimethylsilane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 156 – 186 g; females: 134 – 156 g
- Housing: groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice (Altromin Spezial-futter GmbH & Co. KG, Lage, Germany) provided ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 provided ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Drinking water, municipal residue control, microbiological controls at regular intervals;
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and de-acidified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air. Based on the results of stability testing (Eurofins Munich Study No. 188370), the test item formulations were prepared within the stability time frame (7 days at roomtemperature). The prepared formulation was stored protected from light and at room temperature under an argon layer. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the study monitor based on the test item’s characteristics.
Corn oil was filtered through a mixture of activated silica gel 60 and aluminium oxide (1:1, volume/volume), which had been filled into a glass chromatography column to three quarters of its height. For filtering, a vacuum of 75 mbar was applied. The dried and de-acidified vehicle was overlaid with argon and stored until usage.
- Concentration in vehicle: 25, 62.5, 100, 187.5 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: MKCG3257
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins
Munich Study No. 188370).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of HD and LD groups.
As the test item was shown to be homogenous according to Eurofins Study No. 188370 (after 30 min without stirring), samples were not collected during this study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total).
The mean recoveries observed for the LD dose group was between 89.0 % and 96.4 % of the nominal value, between 91.1% and 95.4% for the MD dose group, between 91.9% and 95.6% for the MID dose group and between 93.1% and 98.3% of the nominal value for HD dose group. The mean recoveries observed in the LD, MD, MID and HD groups were 91.5%, 93.0%, 94.2% and 95% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%.
Duration of treatment / exposure:
90 days
Animals in the recovery group were observed for a period of 28 days following the last administration.
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose (LD)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
medium dose (MD)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
medium intermediate dose (MID)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
high dose (HD)
No. of animals per sex per dose:
main study: 10 per sex per dose
recovery: 5 per sex per dose (control and HD group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the findings of the OECD 422 with the registered substance. 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 a NOAEL. In the above mentioned OECD 422 study, doses of 50 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day were tested. As described in the conclusion of this study, the NOAEL was determined to be 250 mg/kg bw/day based on the results observed at 1000 mg/kg bw/day in male (hepatic protoporphyrin accumulation, adrenal cortical atrophy, kidney protein droplet nephropathy, testicular seminiferous tubule degeneration with epididymides involvement) and in female rats (periportal vacuolation).
- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for general clinical observations, twice daily for morbidity and mortality except for weekends and public holdiays, then only once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter
- Detailed cage side observations (outside home cage in a standard arena) included: 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

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

FOOD CONSUMPTION:
- Food consumption: measured weekly during the treatment and recovery period

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 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
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period as part of the sacrifice of the animals
- Animals fasted: Yes
- How many animals: all
- Parameters examined incl. hormones: 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), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), glucose (Gluc), sodium (Na), potassium (K), T3, T4, TSH, testosterone, FSH, LH, estradiol

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery period as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters examined: specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythrocytes (Ery), leukocytes (Leuc), urine colour and appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 1)
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119) were sacrificed using anaesthesia (ketamine/xylazin)
and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Detailed gross necropsy was also performed on animals found dead or euthanised for animal welfare reasons during the administration period.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.

HISTOPATHOLOGY: Yes (see Table 1)
The listed organs (Table 1) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the control and HD group sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice. For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
For organs and tissues showing treatment-related changes (i.e. liver, kidney, testes, epididymides, adrenal glands, spleen, bone marrow, thymus) in the HD group, these examinations were extended to animals of all other dose groups as well as to animals
subjected to necropsy at the end of the recovery period. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

ORGAN WEIGHTS: Yes
The wet weight of the following organs of all sacrificed animals was recorded as soon as possible: liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/ parathyroid glands (were weighed after fixation), testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart
Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Other examinations:
Fertility parameters:
In the last two weeks of treatment, the estrous cycle of all female animals was monitored. In all female recovery animals the estrous cycle was also monitored at least during the last week of the recovery period.
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser (TOX IVOS Version 13.0).
Sperm morphology slides were prepared from all male animals (main and recovery).
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 will be performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics will be performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only clinical sign observed in the two moribund animals was moving the bedding (100 mg/kg bw/day female no. 72 on treatment days 1-6; 250 mg/kg bw/day female no. 88 on treatment days 9-12). No other clinical findings were recorded prior to the gavage error which resulted in the deaths of these animals.
No clinical findings were observed in males of the control group during the treatment period. Exophthalmos of the left eye observed in female control animal no. 68 on study days 89 and 90 was considered to be an incidental finding. No other clinical findings were observed in female control animals.
Moderately or slightly reduced spontaneous activity was seen in male no. 42 on day 25, male no. 45 on days 26-28, and male no. 56 on days 40-47, all in the 750 mg/kg bw/day group. Slightly reduced spontaneous activity was additionally noted for two 400 mg/kg bw/day females (nos. 91 and 92) and two 750 mg/kg bw/day females (nos. 101 and 102) on 6 days in the first week of the treatment period.
For female no. 75 of the 100 mg/kg bw/day group and female no. 103 of the 750 mg/kg bw/day group diarrhoea was observed on treatment day 7-8 or day 40. This is considered to be incidental and not an adverse systemic effect of the test item.
Slight to severe salivation and/or moving the bedding was found on several treatment days for all male animals in the 100 mg/kg bw/day group within the first week of the treatment period, within the first half of the treatment period for all male animals in the 250, 400 and 750 mg/kg bw/day groups and additionally for single animals in the 750 mg/kg bw/day group until the end of the treatment period. For three 100 mg/kg bw/day females (animal nos. 72, 73 and 74) moving the bedding was noted on days 1-6, slightly increased salivation in one 250 mg/kg bw/day female (animal no. 82) and moving the bedding for all animals in the female 250 mg/kg bw/day group were observed during the first two weeks of the treatment period. In the female 400 mg/kg bw/day group, slight to severe salivation and/or moving the bedding was found for all animals within the first three weeks of treatment and salivation for two animals additionally on one day of the treatment (day 68, 400 mg/kg bw/day female nos. 92 and 93). In the female 750 mg/kg bw/day group, moving the bedding and/or salivation at a slight to severe degree was found in all animals within the first half of treatment period and for single animals on several days additionally in the second half of the treatment period. The clinical signs salivation and moving the bedding were observed in timely relation to the dose administration and are therefore considered to be signs of a local reaction to or a bad taste of the test item rather than a toxicological relevant effect.
Moderate salivation observed for 750 mg/kg bw/day female no. 118 on day 91 during the recovery period is considered to be an incidental finding or possibly a conditioned response. No clinical findings were seen in any other male or female animals during the recovery period on days 91 to 118.

Detailed clinical observation in males showed statistical significance for an increased number of animals showing salivation in week 2 in the 250 mg/kg bw/day and 400 mg/kg bw/day groups (45% and 55 % above control, respectively) and in the 750 mg/kg bw/day group in week 4, for a decrease in animals found sleeping in the 750 mg/kg bw/day group (46.7% below control) and consequently an increased number of animals moving in the cage when compared to controls, for an increased number of animals showing salivation in week 8 in the 750 mg/kg bw/day group (13.3% above control) and in week 11 a decreased number of animals sleeping (33.3% below control) and an increased number of animals moving in the cage in the male 750 mg/kg bw/day group.
No statistical significances were found in all female dose groups when compared to the control group until study week 9. In week 9, the number of animals showing salivation was found increased by 12.5%, in week 11 by 40% and in week 12 by 16.7% when compared to controls in the 750 mg/kg bw/day group. Response to handling increased with statistical significance in week 11 in females of the 750 mg/kg bw/day group (25.0% above control).
In male and female recovery groups, no statistical significances were noted for detailed clinical observation for the males and female 750 mg/kg bw/day group during the recovery period.
The statistical significances were seen occasionally in single weeks in the dose groups and therefore are considered not to be an adverse effect of the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from 100 mg/kg bw/day group (no. 72) was euthanized on study day 55 and one female from 250 mg/kg bw/day group (no. 88) was found dead on day 20 of the treatment period. The cause of death was found to be gavage error in both animals, leading to a trauma. Therefore, the death was not caused by a systemic effect of the test item.
All remaining animals survived until the end of the treatment period or the recovery period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The treatment with the test item had an effect on body weight development in the male 400 and 750 mg/kg bw/day and female 750 mg/kg bw/day group with statistically significant changes when compared to the respective control group.

The mean body weight of all male and female dose groups and the control group increased during the treatment period (day 1 compared to day 90), but was found with a statistical significant decrease in the male 400 mg/kg bw/day and 750 mg/kg bw/day group starting from day 15 to day 90 compared to the control group. Mean body weight in males was statistically significantly decreased between 5.39% and 14.76% when compared to control for the 400 mg/kg bw/day group and between 5.70% and 19.53% below control for the male 750 mg/kg bw/day group. In females, the mean body weight was statistically significantly decreased on day 57 (5.86% below control) and 5.46% below control on day 78 in the 750 mg/kg bw/day group. At the end of the treatment period on day 90 the body weight was statistically significantly decreased in the male 400 mg/kg bw/day and 750 mg/kg bw/day dose groups (14.54% and 19.22% below control) and in the female 250 mg/kg bw/day, 400 mg/kg bw/day and 750 mg/kg bw/day group (6.23%, 6.15% and 7.69% below control).
During the recovery period, mean body weight in males showed a decrease with statistical significance in the 750 mg/kg bw/day group between 19.58% below control on day 90 and 12.81% on day 118, but the group mean weight increased between day 90 compared to day 118 in the male and female control and 750 mg/kg bw/day group. No statistical significance was found at the end of the recovery period for the female 750 mg/kg bw/day group.
The overall body weight change between day 1 and day 90 of the treatment period was seen with a dose dependent and statistical significant decrease for the male dose groups. The change was statistically significant in the male 400 mg/kg bw/day and 750 mg/kg bw/day group (30.81% and 40.99% below control). In females, the overall body weight change between day 1 and day 90 was statistically significantly 21.99% below control in the 250 mg/kg bw/day group, 21.03% below control in the 400 mg/kg bw/day group and 28.83% below control in the 750 mg/kg bw/day group.
The weekly change during the treatment period showed statistical significance in the male 750 mg/kg bw/day group in week 2 to week 6 (up to 56.6% below control in week 6) and in week 10. In the male 400 mg/kg bw/day group the change was significantly below control in week 1 (20.1%), in week 6 (43.4%) and in the male 100 mg/kg bw/day group 49.2% below control in week 6. An increased change with statistical significance was found in the female 100 mg/kg bw/day group in week 5 (294.3% above control).
An overall mean body weight change in the recovery period between day 90 and 118, was statistically significant in males of the 750 mg/kg bw group (84.8% above control) and significantly increased in week 1 compared to the controls. In females, the decreased change of 80.0% between day 104 and day 111 was statistically significant when compared to controls, but the overall change (day 90 to day 118) was increased (7.8% above control) without statistical significance. Thus, the decrease in body weight gain during the treatment period showed a tendency to recover during the recovery period when compared to control.
The statistical significances in the female 250 mg/kg bw/day and 400 mg/kg bw/day group were considered not to be of an adverse effect of the test item, as no specific changes were seen during the entire treatment period and no dose dependent changes were noted between these groups. The single statistical significant difference in the male 100 mg/kg bw/day group was considered not to be related to treatment with test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males of the 750 mg/kg bw/day group there was statistically significantly lower food consumption in week 4 and from week 6 until the end of the treatment period, when compared to controls. Food consumption was statistically significantly decreased between 16.97% and 53.97% below control in males. In the female 750 mg/kg bw/day group, statistical significant reduction of food consumption was noted between study weeks 2 and 12 (between 8.32% and 22.59% below controls). The minimum number of individual values for statistical evaluation is n=3. Therefore, a statistical evaluation of the 100, 250 and 400 mg/kg bw/day group was not performed.
During the overall treatment period, the mean total food consumption was lower in all male and female dose groups when compared to the control group. The reduction was between 16.86% (100 mg/kg bw/day group) and 27.87% (highest reduction in the 400 mg/kg bw/day group) in males and between 7.12% (100 mg/kg bw/day group) and 16.09% (highest reduction in the 400 mg/kg bw/day group) in females.
The weekly mean food consumption in the male and female 750 mg/kg bw/day group during the recovery phase was comparable to the mean consumption noted for the male and female control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment or recovery phase no adverse test item-related effect on haematological parameters was found. No test item-related effect on coagulation parameters was considered.
Slight but statistically significantly increased RBC count was observed in males of the 750 mg/kg bw/day group (5.35% above control), slightly decreased MCV in the male 400 mg/kg bw/day and 750 mg/kg bw/day group (6.96% and 7.96% below control) and slightly decreased MCH in the male 250, 400 and 750 mg/kg bw/day group (between 3.28% and 8.61% below control) were observed. In females mean MCV was also slightly and statistically significantly decreased in the 750 mg/kg bw/day group (MCV: 7.42% below control) and MCH in the 250, 400 and 750 mg/kg bw/day group (between 4.10% and 8.54% below control).
A statistically significantly lower reticulocyte rate was seen in the male 250, 400 and 750 mg/kg bw/day group, when compared to control (between 18.12% (250 mg/kg bw/day) and 43.09% (250 mg/kg bw/day)), but no dose dependency was found. In females, a decrease in reticulocytes of 21.74% compared to control was seen in the 750 mg/kg bw/day group without statistical significance.
As the changes on the red blood cell parameters were slight and mean group values were comparable to the respective control group, an adverse test item-related effect is not considered.
In the differential blood cell count, mean monocyte count was statistically significantly increased in the male 400 mg/kg bw/day group (66.30% above control) and the 750 mg/kg bw/day group (86.90% above control), whereas the increase of 34.44% in the 250 mg/kg bw/day group was without statistical significance. The basophile count showed a statistical significant increase of 214.81% above control in the male 750 mg/kg bw/day group. In all other male dose groups the mean values for basophiles were above control, but no dose dependency was seen.
A statistically significant increase in lymphocytes in females of the 250 mg/kg bw/day group was seen (9.57% above controls) and in neutrophils a decrease for all dose groups (between 25.08% above control in the 400 mg/kg bw/day group and 42.61% in the 720 mg/kg bw/day group). Regarding monocytes, an increase with statistical significance was observed in females in the 750 mg/kg bw/day group (142.70% above control). A slight increase without statistical significance was also seen in the 400 mg/kg bw/day group (43.16% above control).
The slight changes in differential blood cell count do not indicate acute or chronic inflammation and are considered not to be an adverse effect of treatment with the test item.
At the end of the recovery period in 750 mg/kg bw/day animals, slightly statistically significantly decreased values were found for MCV (4.67% below control), MCH (8.29% below control) and MCHC (3.96% below control) and a slight increase in PLT of 15.24% above control. Overall, the mean values were comparable to the control group. The decrease of mean reticulocytes observed at the end of treatment recovered at the end of the recovery phase when the mean value was increased by 29.19%. At the end of the recovery period, the mean basophiles count recovered and showed a decreased mean value (20.00% below control) without statistical significance.
In females, statistical significances were noted at the end of the recovery period for a slight increase of 6.12% above control for the mean RBC and a slight decrease of mean MCH (6.39% below control). No further statistical significances were found in females for all other parameters and mean values were comparable to control.
Mean coagulation parameters at the end of the treatment period were minimally and statistically significantly increased for aPTT in the male 400 mg/kg bw/day group (23.62% above control) and 750 mg/kg bw/day group (33.26% above control) and in females, for PT in the female 100, 250 and 750 mg/kg bw/day group (between 6.92% and 8.54% above control) and for aPTT in the 400 and 750 mg/kg bw/day group (20.70% and 19.68% above control). No statistical significant changes were noted in the male and female recovery group. As the statistical significant changes for aPTT were seen without dose dependency or were seen only in females (PT) and values were within the range of historical control data an adverse test item-related effect on coagulation parameters is not considered in this study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment or recovery phase no adverse test item-related effect on clinical biochemistry parameters was found.
A slightly increased K level was seen in male animals of the 750 mg/kg bw/day group (34% above control) and female animals of the 400 and 750 mg/kg bw/day groups (28% and 52% above control) at the end of the treatment period. In females – but not males serum K level was also slightly increased at the end of the recovery period (26% above controls). However, no adverse histopathological findings for kidneys were seen. Therefore, the statistical significance of K is considered to be of no toxicological relevance.
Slightly but statistically significant lower serum AP (in male 250, 400 and 750 mg/kg bw/day groups between 31% and 32% below control and female 750 mg/kg bw/day group 54% below controls), serum TBil (in male 750 mg/kg bw/day group 43% below controls) and serum Crea (in female 250 and 400 mg/kg bw/day groups 19% and 21% below controls, respectively) levels were found in dose groups, when compared to controls at the end of the treatment period. The levels were within the range of historical control data and as they were only marginally different this is considered to be without toxicological relevance. Slightly but statistically significant higher Chol (32% above controls), TBA (118% above controls) and HDL (34% above controls) in females of the 250 mg/kg bw/day group – but not 750 mg/kg bw/day group is considered biologically not relevant.
The statistical significant decrease of 24.67% compared to control for Gluc in the male recovery group, the increase of ASAT in the female recovery group (55.93% above control), the increase of urea (17.64% above control) and LDL 60.98% above control) in the female recovery group were considered not to be of toxicological relevance as no such effect was seen at the end of the treatment period.
Hormone analysis in serum samples did not show an effect related to the treatment with test item at the end of the treatment and recovery period for all hormone parameters measured in the study (T3, T4, TSH, testosterone, FSH, LH, estradiol).
At the end of the treatment period statistical significant lower serum T3 level (31.8% below control) was noted in the males of the 750 mg/kg bw/day group and an increase of 185% for testosterone in the 400 mg/kg bw/day group – but not 750 mg/kg bw/day group. Besides, there were no statistical significances for all hormone parameters (T3, T4, TSH, testosterone, FSH, LH, estradiol) in the female dose groups at the end of the treatment period and no dose dependency was seen in both genders. For males and females no statistical significant changes or considerable differences to the control group were noted at the end of the recovery period.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis in the male and female animals showed the presence of bilirubin in single animals of all male dose groups and in a single animal in the female 400 mg/kg bw/day group (animal no. 97) but not in the control group. As the slight changes noted for bilirubin at clinical biochemical investigation are considered to be of no toxicological relevance, the presence of bilirubin is considered to be not test item-related.
All other parameters measured at the end of the treatment and recovery periods were without specific changes to the respective control group and no test item-related effects on urinary parameters were found.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Before the first treatment, statistically significantly fewer male animals were found sleeping in the cage (down to 88.5% of controls). An increased number of animals were moving in the cage in all dose groups (575% above control), an increase of response to handling in the 400 mg/kg bw/day group (12.5% above control) and 750 mg/kg bw/day group (10% above control), an increased response to finger approach in the 400 mg/kg bw/day group (10% above control), an increase in the number of supported rearings in the 400 mg/kg bw/day group (204.8% above control), an increase in animals showing defecation of 333.3% above control in the 750 mg/kg bw/day group, equilibrium reflex in the 400 mg/kg bw/day group of 10% above control, and an increase of body temperature of 2.51% in the 400 mg/kg bw/day group and 2.31% above control in the 750 mg/kg bw/day group were also observed. No statistical significances were noted in the last week of treatment (week 13) for any male dose group.
In females, defecation was seen in statistically significantly more animals in the week before the start of treatment in the 400 mg/kg bw/day and 750 mg/kg bw/day groups. No further statistical significances were noted for females in the week before treatment or in the last week of the treatment period.
At the end of the recovery period, statistically significantly more 750 mg/kg bw/day females were seen sleeping and 60.0% fewer animals than in the control group were moving in the cage. The body temperature of the female 750 mg/kg bw/day recovery group was statistically significantly increased when compared to control (1.60% above control). No statistical significances were seen in the male 750 mg/kg bw/day recovery group.
As the statistical significances were seen before start of treatment or at the end of the recovery period in the females of the 750 mg/kg bw/day group but not in males and additionally, no clinical findings were noted during the recovery period in both genders, no adverse effect of the test item is considered.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weight of brain in males was statistically significantly decreased in the 250 mg/kg bw/day, 400 mg/kg bw/day and 750 mg/kg bw/day groups (between 4.6% and 7.5% below control) and in females (6.4% in the 750 mg/kg bw/day group and 5.2% in the 400 mg/kg bw/day group, when compared to control). In relation to body weight, however, an increase in the male 400 mg/kg bw/day and 750 mg/kg bw/day groups of up to 14.4% with statistical significance was noted. This is related to a statistically significantly reduced final body weight in males of 400 mg/kg bw/day and 750 mg/kg bw/day groups.
The weight of the spleen showed a tendency towards a decrease in the male and female dose groups and was statistically significantly decreased in the male 750 mg/kg bw/day group (absolute 17.2% below control) and female 400 mg/kg bw/day and 750 mg/kg bw/day groups (absolute 19.4% and 15.9% below control). The relative organ weight to brain or body weight was seen without dose dependency in females and no statistical significances were noted for the relative weight.
Furthermore, weight of thymus showed a statistical significant decrease in the male 400 mg/kg bw/day and 750 mg/kg bw/day groups and the female 750 mg/kg bw/day group. Absolute weight in males was up to 42.8% below control in males (400 mg/kg bw/day group) and 29.3% below control in the female 750 mg/kg bw/day group. Relative weight was up to 39.4% below controls in males (400 mg/kg bw/day group, relative to brain weight) and 24.7% below controls in females (750 mg/kg bw/day group, relative to brain). Histopathologically a dose-dependent increase of incidence and/or severity of thymic atrophy was noted and is considered to be a stress-related finding.
Organ weight of thyroid/parathyroid gland was statistically significantly increased in the male 750 mg/kg bw/day group (relative to body weight 22.2% above control). No dose dependency was noted between the male or female dose groups. This did not coincide with histopathological findings or changes in serum thyroid hormone levels. No test itemrelated effect is therefore considered.
Statistically significant changes were noted for the decreased absolute weight of kidney in the male 400 mg/kg bw/day group (13.7% below control) and 750 mg/kg bw/day group (11.9% below control). No statistically significant changes were seen in the female dose groups. As no dose dependency was noted and histopathological evaluation showed no test item-related findings, the statistically significant decrease of the absolute kidney weight in the male dose groups is considered to be of no toxicological relevance.
Considering the weight of the pituitary glands, there were statistically significant changes in the male 400 mg/kg bw/day and 750 mg/kg bw/day groups, but not in the female dose groups. An absolute decrease of up to 24.6 % below control was found in the 750 mg/kg bw/day group and up to 18.8% below control, relative to brain weight for the 400 mg/kg bw/day group. As no such effect was seen in the female dose groups and histopathological evaluation showed no adverse findings, no toxicological effect is considered.
Mean absolute weight of heart in males was statistically significantly decreased in the male 400 mg/kg bw/day group (15.4 % below control) and the 750 mg/kg bw/day group (15.6% below control). In the female dose groups no absolute or relative decrease was noted. Mean relative heart weight was statistically significantly increased in the female 250 mg/kg bw/day group and 750 mg/kg bw/day group (12.5% and 13.1% above control). Due to the inconsistency in organ weight changes between the male and female dose groups and the absence of histopathological findings, no test item-related effect is considered.
A marked and statistically significant increase of the absolute and relative liver weight was noted in the male and female dose groups (250 mg/kg bw/day, 400 mg/kg bw/day and 750 mg/kg bw/day group). In males, absolute weight showed an increase of 20.6% in the 750 mg/kg bw/day group. In females the increase was between 16.5% and 49.4% in the 250 mg/kg bw/day, 400 mg/kg bw/day and 750 mg/kg bw/day groups. Relative liver weight to body weight or brain weight was statistically significantly increased in the male and female 250 mg/kg bw/day, 400 mg/kg bw/day and 750 mg/kg bw/day groups when compared to controls (males, 750 mg/kg bw/day group: relative to body weight up to 49.7%, relative to brain weight up to 30.3% and females, 750 mg/kg bw/day group: relative to body weight up to 62.4%, relative to brain up to 59.6%). The increase of the liver weight between the dose groups was more prominent in females. At histopathological evaluation a minor severity of hepatocellular hypertrophy was found, which increased in incidence by dose from the 100 mg/kg bw/day group and up.
For the reproductive organs of the male dose groups, a statistical significant weight decrease was noted for testes in the 750 mg/kg bw/day group (absolute weight 40.4% below control, relative to brain weight 35.9% below control). However, testes weight in the 400 mg/kg bw/day group (relative to body weight 17.9% above control) was even slightly and statistically significant higher than in controls and slightly increased relative to brain in the 250 mg/kg bw/day group (12.4% above control). Furthermore, absolute and relative weight of epididymides was statistically significantly decreased in the 750 mg/kg bw/day group (up to 37.1% below control) and additionally the absolute and relative weight of prostate was also decreased (absolute 31.9% and relative to brain 26.4% below control). The decrease in reproductive organ weights correlates with the small size of testes, epididymides and prostate gland/seminal vesicles observed at necropsy.
At the end of the recovery period in the male 750 mg/kg bw/day group a statistically significant decrease was noted for thymus weight (absolute 23.5% below control, relative to brain weight 19.3% below control) and a statistically significant increase of heart weight (relative to body weight 13.2% above control). In the male recovery 750 mg/kg bw/day group statistically significant decreases were seen for testes (absolute 21.7% below control, relative to brain weight 17.1% below control), the absolute and relative epididymides weight to brain weight (absolute 32.9% below control, relative up to 29.01% below control) and for weight of prostate gland with seminal vesicles and coagulating glands (absolute 19.7% below control, relative to brain 15.3% below control). Further statistically significant increases were noted for the kidney weight relative to body weight in the female 750 mg/kg bw/day group (8.9% above control) and the liver (18.1% above control).
The comparison of the statistically significant organ weight changes at the end of the recovery period to the end of the treatment period showed, that the decrease of thymus weight in the male 750 mg/kg bw/day group, the decreased weight of the male reproductive organs and the relative weight of the liver in the female 750 mg/kg bw/day group did not fully recover by the end of the recovery period.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Decedents:
Macroscopic findings observed for the euthanised 100 mg/kg bw/day female on study day 55 were a filled thoracic cavity and nose with red fluid, a red coloured lung and a wound at the tail. The 250 mg/kg bw/day female animal that was found dead on day 20 was seen with a general autolyse at day of necropsy. The cause of death for these prematurely deceased animals was accidentally due to technical reason, leading to a trauma. Therefore, the death of both animals was not caused by a systemic effect of the test item, but accidentally.
Scheduled necropsy:
At the end of the treatment period test item-related macroscopic findings in males were found in the 750 mg/kg bw/day group for reproductive organs. With exception of one 750 mg/kg bw/day male animal, testes and epididymis of all 750 mg/kg bw/day animals were small sized bilaterally. Additionally, the prostate gland and seminal vesicles of two 750 mg/kg bw/day animals were small sized.
Small epididymides were recorded at the end of the recovery period of one male 750 mg/kg bw/day animal and several red foci on the thymus of one 750 mg/kg bw/day female animal.
The liver was found with an enlarged size in nearly all female 750 mg/kg bw/day animals. In addition, findings at the liver of one female 100 mg/kg bw/day animal (enlarged right medial lobe) but also one control animal (enlarged median lobe, reduced left lateral lobe) were recorded. At histopathological evaluation, in animals from the 100 mg/kg bw/day group onwards a minor severity of hepatocellular hypertrophy that dose-dependently increased in incidence was noted.
All other findings are considered to be not realted to the treatemt with the test item as they were seen in single animals of the dose groups or were also found in the control group or no correlation with histopathological findings were noted.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Testes, epididymides, prostate, seminal vesicles:
The testes of all males at 750 mg/kg bw/day and of one animal at 400 mg/kg bw/day were affected by tubular degeneration. Spermatids and spermia were mainly affected. In eight animals at 750 mg/kg bw/day, there was slight to moderate interstitial oedema, and in a few animals from 250 to 750 mg/kg bw/day there was tubular oedema at minor severity degrees.
In the epididymides, the following test item-related secondary lesions were noted: slight detritus in two animals at 750 mg/kg bw/day, increased pyknotic sperm cells in one animal at 400 mg/kg bw/day, and oligospermia in nine animals at 750 mg/kg bw/day.
After the treatment-free recovery period, in the testes of HD animals there were still slight tubular edema in one male, tubular degeneration in three animals and tubular vacuolation (Sertoli’s cells) at a minimal to slight degree. In the epididymides from all animals, there was detritus and oligospermia, and in four animals, there were increased pyknotic sperm cells.
In the prostate gland and seminal vesicles, there was reduced secretion in two main test animals at 750 mg/kg bw/day.
Liver:
In animals from 100 mg/kg bw/day and up, there was a minor severity of hepatocellular hypertrophy that increased in incidence by dose. After the recovery period, no test item-related finding was noted.
Stress-related findings:
In the main test males, the severity of cortical (zona fasciculata) vacuolation increased in animals at 400 and 750 mg/kg bw/day. From 250 mg/kg bw/day and up, there was an increasing incidence of adrenal cortical hypertrophy in females. In single males at 250 mg/kg bw/day, females at 400 mg/kg bw/day, and males at 750 mg/kg bw/day, there was a minor cortical (zona fasciculata) atrophy.
After the recovery period, there was still cortical hypertrophy in a few females at 750 mg/kg bw/day, and atrophy in males at 750 mg/kg bw/day.
One male at 400 mg/kg bw/day was affected by PALS atrophy in the spleen. The thymic atrophy and fatty replacement of the sternal bone marrow increased in incidence and/or severity by dose. These findings recovered.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Fertility parameters:
A test item-related effect on the fertility parameters was found for the male 750, 400 and 250 mg/kg bw/day group.
Testis weight in the 750 mg/kg bw/day group was decreased at the end of treatment and at the end of the recovery period when compared to the control group (38.9% and 23.4% below controls, respectively) and considered to be an effect of the test item. Mean weight of testes in the 100, 250 and 450 mg/kg bw/day group was comparable to the mean weight in the control group.
Testicular sperm count was statistically significantly decreased in the 750 mg/kg bw/day group at the end of the treatment period (65.6% below control) and did not fully recover until the end of the recovery period (36.3% below control). The mean sperm count in all other dose groups was comparable to the control group.
Mean sperm motility parameters were statistically significantly decreased in the 250, 400 and 750 mg/kg bw/day group at the end of the treatment and for the 750 mg/kg bw/day group at the end of the recovery period. In the 400 mg/kg bw/day group, the decrease was 22.0% below control for the mean motile count, 120.7% above control for static and 29.6% below control for the rapid count and 20.2% below for the mean rapid count in the 250 mg/kg bw/day group. Motile count in the 750 mg/kg bw/day group was 92.2% below control, static count 504.8% above control and for rapid count 93.5% below control at the end of the treatment period. These statistical significant differences were also seen at the end of the recovery period. The decreased mean motility of sperms in the 250, 400 and 750 mg/kg bw/day group is considered to be a test item-related effect.
Sperm morphology showed marked and statistical significant changes in the absolute and relative number of normal and abnormal changes in the 250, 400 and 750 mg/kg bw/day group. The relative number of normal sperms was dose dependently decreased (250 mg/kg bw/day 14.3%, 400 mg/kg bw/day 23.4% and 750 mg/kg bw/day 76.2% below control) and abnormal sperm findings were increased (250 mg/kg bw/day 321.2%, 400 mg/kg bw/day 528.2% and 750 mg/kg bw/day 1717.6% above control). Abnormality of sperms concerned mainly the finding of separated heads and tails, which were additionally found broken or coiled. The statistical significant alterations were not recovered at the end of the recovery period and changes were comparable to the end of the treatment period.
There was a statistically significantly decreased number of normal estrous cycles in the 400 and 750 mg/kg bw/day group females when compared to controls at the end of the treatment period. At the end of the recovery period, the statistically significant decrease for the number of normal cycle did not recover. No correlating findings were noted at histopathological assessment for the female reproductive organs and no abnormalities in hormone analysis were seen. Therefore, the statistically significant decrease was considered not to be an adverse effect of the test item.
The statistically significant increase of mean cycle length at the end of the recovery period is considered to be incidental as no statistical significance was found at the end of the treatment period.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced sperm motility, effects on sperm morphology and tubular edema at 250 mg/kg bw/day

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this oral 90-day repeated dose toxicity study in rats the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg bw/day based on the following findings at 250 mg/kg bw/day: reduced male and female body weights, reduced sperm motility, effects on sperm morphology as well as tubular edema.