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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Mar - 28 Jul 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

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
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[3-(trimethoxysilyl)propyl]butylamine
EC Number:
250-437-8
EC Name:
N-[3-(trimethoxysilyl)propyl]butylamine
Cas Number:
31024-56-3
Molecular formula:
C10H25NO3Si
IUPAC Name:
butyl[3-(trimethoxysilyl)propyl]amine
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. 6-7 weeks
- Weight at study initiation: males: 159 – 194 g (mean: 174.44 g, ± 20 % = 139.55 – 209.33 g); females: 140 – 170 g (mean: 152.28 g, ± 20 % = 121.82 – 182.74 g)
- Fasting period before study: no
- Housing: groups of 5 animals/sex/group/cage in IVC cages (type IV, polysulsphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food and water are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH; no known contaminants in the feed or water at levels that would interfere with the objectives of the study were present.

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 corn oil was dried and de-acidified as follows: Corn oil was filtered through a mixture of activated silica gel 60 and aluminum oxide (1:1, volume/volume), which has 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.
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 (w/w). The formulation was alternately 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. Formulates were kept under magnetic stirring during the daily administration.
Based on the results of stability testing (Eurofins Munich Study No. 192943), the test item formulations were prepared within the stability time frame (2 days at room temperature). The prepared formulation was stored protected from light and at room temperature.


VEHICLE
- Justification for use and choice of vehicle: selected in consultation with sponsor based on test substance characteristics
- Concentration in vehicle: 62.5/25, 125/75, 250/187.5/125 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: MKCK6411
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 as part of a separate GLP study (Eurofins Munich Study No. 192943).
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was not shown to be homogenous according to Eurofins Study No. 192943, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1, 5, 9 and in the last week of treatment (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 192944) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below -15 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Concentration analysis:
The concentration of formulation samples in dried and deacidified corn oil was determined at three concentrations, 62.5 mg/mL, 125 mg/mL and 250 mg/mL in study week 1. After week 1 of the main study, the concentrations of the applied dosing solutions were reduced to 25 mg/mL, 75 mg/mL and 125 mg/mL for the rest of the study. After the reduction of the concentrations the LD samples were out of the validated range and were therefore, below the limit of quantitation (BLQ). The mean recoveries observed for week 1 was 92.4% of the nominal value for LD group, 93.9% of the nominal value for MD group and 100.6% of the nominal value for HD group samples. The mean recoveries during the rest of the study in weeks 5, 9 and the last week could not be established for the LD group, which was BLQ. For the MD, mean recoveries varied between 83.1% and 89.2% of the nominal value and for the HD, mean recoveries varied between 84.9% and 91.3% of the nominal value. The overall mean recoveries determined in the MD and HD samples were 86.5%, and 87.4% of the nominal concentration. Overall mean recoveries for the LD samples could not be established as they were BLQ after the concentration reduction.
Nominal concentrations were confirmed for all dose groups, but measured concentrations for the MD and HD groups were slightly outside the acceptance criteria of 10%. This had no influence on the validity of the study.

Homogeneity:
Homogeneity of formulation samples was determined in study weeks 1, 3, 5 and the last week of the study.
The coefficients of variation of the different sampling locations (top, middle, bottom) was 1.6% in LD group in week 1, between 0.4% and 2.6% in MD dose group and between 0.9% and 2.1% in HD dose group. All samples were homogenous, as CV was below or equal 10%.
Duration of treatment / exposure:
90 days (control and test groups)
90 days and 28 days recovery period (satellite control and high dose group)
Frequency of treatment:
one daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
dose was reduced on Day 10 to 100 mg/kg bw/day until the end of the study (low dose, LD)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
dose was reduced on Day 10 to 300 mg/kg bw/day until the end of the study (medium dose, MD)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
dose was reduced on Day 10 to 750 mg/kg bw/day and again on Day 26 to 500 mg/kg bw/day until the end of the study (high dose, HD)
No. of animals per sex per dose:
10 (main study)
5 (satellite control and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on a 14-day dose range finder study of the test item (BSL Munich Study No. 192938). In the dose range finder study the doses were 200, 500 and 750 mg/kg bw/day. In the dose range finder study no mortality occurred in the control or any of the dose groups during the treatment period of this study with the exception of two female animals in the mid dose group. One animal was found dead and one animal was sacrificed in moribund condition. In both animals it was not considered to be caused by the test item. There were no test item related clinical signs of systemic toxicity observed during the treatment period in any of the animals. No test item related effect on body weight and food consumption was observed in males and females. No test item related effect on haematology, clinical chemistry parameters and organ weight was observed. No treatment related macroscopic findings were observed in any male or female dose groups at necropsy.
The highest dose level is chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels is selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group will be handled in an identical manner to the test group subjects and will receive the vehicle using the same volume as used for the high dose group.

- Fasting period before blood sampling for clinical biochemistry: over night fasting
- Rationale for selecting satellite groups: examine recovery
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day general clinical observation, preferably at the same time each day and considering the peak period of anticipated effects after dosing; twice daily for morbidity and mortality except on weekends and public holidays when observations were made once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and at least once a week thereafter
- Observations checked outside the home cage in a standard arena: response to handling, body position, spontaneous locomotor activity, ataxic gait, hypotonic gait, twitches, tremors, seizures, unusual behaviour, stereotypical behaviour, faeces consistency, abnormal vocalization, aggressiveness/irritability and grooming

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

FOOD CONSUMPTION: YES
- Time schedule for examinations: weekly during the treatment and recovery period

FOOD EFFICIENCY: No

WATER CONSUMPTION: 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
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment and recovery period as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked: 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), monocytes (Mono), eosinophils (Eos), lymphocytes (Lym), 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 treatment and recovery period
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked: 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)

SERUM HORMONES: Yes
- Time of blood sample collection: at the end of the treatment and recovery period
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: T3, T4, TSH

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

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and towards the end of the exposure period but not earlier than in Week 11 as well as in the last week of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: The FOB tests included 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

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: Careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
The following organ weights were determined: liver; prostate, seminal vesicles and coagulating glands; spleen; kidneys; ovaries; brain; adrenals; uterus with cervix; pituitary gland; testes; thymus; heart; epididymides; thyroid/ parathyroid glands (weighed after fixation)

HISTOPATHOLOGY: Yes: adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar & cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland (only if changes were observed during ophthalmological examinations), heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular, parotis), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland including parathyroid glands, trachea, ureters, urinary bladder, uterus with cervix and vagina, larynx, head with paranasal sinuses (flushed with fixative)
Other examinations:
Fertility parameters: Daily over a period of 8 days, the oestrous cycle of all female animals were monitored in the last two weeks of treatment. In the recovery animals the oestrous cycle was also monitored during the last weeks of the recovery period. In all male animals, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters on the day of necropsy.
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).
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.
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 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 were 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):
Slight to severe salivation was noted in 2/15 males of the control, 5/10 males and 3/10 females of the MD group and in 11/15 males and 6/15 females of the HD group. Furthermore, moving the bedding was noted in 1/10 males of the LD group, in 6/10 males and 2/10 females of the MD group and in 8/15 males and in 4/15 females of the HD group.
These clinical signs of moving the bedding and salivation were observed immediately after the dose administration and therefore were considered to be signs of a local reaction, typically irritation due to the test item rather than a systemic adverse effect, which was deemed to have no toxicological relevance.
Other clinical findings such as abnormal breathing and piloerection were seen in some male and female animals of the MD and HD groups and in some female animals of the LD group mostly before the dose reduction. Decedent animals showed clinical signs such as spontaneous activity reduced, abnormal breathing, piloerection, half eyelid closure, nasal discharge, wasp waist, coughing/sneezing, slow movements, hunched posture, dehydration, pale skin, hypothermia and diarrhoea before their death, which were observed on several or single days in most of the animals. These signs are considered to have arisen in relation to the gavage method applied for oral dosing; that is, the regurgitation or gastroesophageal reflux of the test item formulation to the upper respiratory tracts and subsequent aspiration and possible irritative physicochemical properties of the test item formulation as determined during the histopathological analysis.
Clinical symptoms like hairless area, crust, scratch/cut and chromodacryorrhea were observed mostly transiently or on single days and within the normal background frequency. One control male and one HD male had half eyelid closure on single days and one MD male had a wasp waist on single days. These symptoms were not considered to be test item-related.
No clinical symptoms were observed during the recovery period.
No statistically significant effects on detailed clinical examination parameters were noted.
Mortality:
mortality observed, treatment-related
Description (incidence):
Under the conditions of this study, there were 20 premature deaths. In the MD group, one male (animal no. 21 on study day 15) and three females (animal no. 74 on study day 4, animal no. 75 on study day 2 and animal no. 80 on study day 75) were found dead, and two males (animal no. 24 on study day 7 and animal no. 25 on study day 60) were sacrificed in moribund conditions. In the HD group, three females died (animal no. 82 on study day 5, animal no. 84 on study day 6 and animal no. 89 on study day 4), and eight males (animal no. 31 on study day 9, animal no. 32 on study day 25, animal no. 33 on study day 8, animal no. 36 on study day 22, animal no. 37 on study day 16, animal no. 40 on study day 21, animal no. 46 on study day 7 and animal no. 47 on study day 29) and three females (animal no. 85 on study day 5, animal no. 86 on study day 12 and animal no 99 on study day 6) were sacrificed in moribund conditions. In addition, animal no. 38 was sacrificed in moribund condition on study day 2 and replaced with animal no. 101.
All other animals survived their scheduled study period. No further mortalities were observed during the recovery period.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
The test item had no effect on body weight development in this study. Overall, the mean body weight increased normally during the treatment and recovery periods in control as well as in dose group animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The test item had no effect on food consumption in this study. Mean daily food intake of male and female animals was in the normal range of variation throughout the treatment and recovery period of this study and without considerable differences between the groups. Slight but statistically significant increase was noted in male HD group in study week 8, 9 and 11 and in female HD group in study week 12 when compared to control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopy examination did not reveal any test item-related effects in any of the treatment groups during the treatment period and during the recovery phase.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No biologically or statistically significant differences in any of the analyzed hematological parameters were found between dose groups and control groups of both male and female animals in this study, with exception of a minimal but statistically significant increase of red blood cell counts in the LD and MD females (~6% and ~5% above controls, respectively).
No biologically or statistically significant differences in any of the analyzed haematological parameters were found in the recovery group.
As the respective values were within the range of historical control data, these changes were not deemed to be toxicologically relevant, but rather consistent with the background variability observed within this laboratory.
At the end of the treatment period and after the recovery period, no statistically significant changes were recorded in blood coagulation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, a statistically significant decrease in TP and TBA levels was found in MD males compared to controls (~6% and 63 % below control, respectively) and in albumin in HD males compared to controls (~5% below control). In females, a statistically significant change was measured for ALAT in the LD group (~34% above control), in glucose in the HD group (~30% below control) and in urea in the LD group (~19% above control). As the mentioned statistical significances occurred only in one gender, a toxicological effect of the test item at the end of treatment could not be determined. Statistically significant changes were only noted for glucose (~30 % below control) and TG (~38 % below control) levels in the females of the HD group after the recovery period. As the mentioned statistical significances occurred only in one gender, a toxicological effect of the test item at the end of treatment could not be determined.
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T3, T4 and TSH, cervix histopathology, coagulating gland histopathology, epididymis histopathology, epididymis weight, estrus cyclicity, liver weight, mammary gland histopathology (males and females), ovary histopathology, ovary weight, oviduct histopathology, prostate histopathology, prostate weight, seminal vesicles histopathology, seminal vesicles weight, sperm morphology, sperm motility, sperm count, testis histopathology, testis weight, thyroid histopathology, thyroid weight, uterus histopathology, uterus weight, vagina histopathology, vaginal smears, adrenals histopathology, adrenals weight, brain weight, pituitary histopathology and pituitary weight. For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The urinalysis of animals sacrificed at the end of the treatment group revealed no test item-related effects and all urinary parameters were within the normal range of variation.
At the end of the recovery period no specific differences were seen in the male HD and female HD groups compared to control groups except for slightly elevated levels of Ery, Protein, and Leuc levels in HD males. The appearance of the urine sample in 2 of the 8 male HD animals was hazy. As there were no histopathological observations in the urinary tract, these findings are not considered to be test item-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In both males and females, no toxicologically relevant effects were observed in any of the parameters of the functional observational battery before and at the end of the treatment or recovery period when compared to controls.
Before the start of the treatment (week -1) in males of the HD group a small but statistically significant increase in body temperature (1.339°C above control), in week 13 in males in the LD and MD groups an increase in the parameter defaecation, in males in the MD group a small but significant increase in vocalization and in females in the HD group a slight but significant reduction in spontaneous activity was observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no body or organ weight changes that could be related to the treatment with the test item.
A statistically significant higher mean thymus weight (~22% above control) was seen in MD female animals. A statistically significant lower mean uterus with cervix weight, mean organ to body weight ratio and mean organ to brain ration (~39% and ~42%, respectively, below control) was seen in female MD animals. Statistically significant higher mean spleen weight was seen for the HD group in female animals (~16% above control).
At the end of the recovery period a statistically significant higher mean epididymides weight (~23% above control) and a statistically significant lower mean spleen to brain weight ratio was seen in HD male animals (~ 13% below control) and a statistically significant higher mean kidney weight and mean organ to body weight ratio (~9% and ~12% above control, respectively) was seen in female HD animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No macroscopic changes that could be due to adverse toxicological systemic effects of the test item were observed in any of the animals examined in this study.
Macroscopic examination of the animals at necropsy listed in female animal no. 51 (C group) abnormal white spotted colour of the lung, in female animals no. 53 (C group) complete dilatation of the uterus, in female animal no. 58 (C group) a completely enlarged pituitary gland, in male animal no. 12 (LD group) an abnormal red spotted thymus, in male animal no. 18 (LD group) enlarged left mandibular lymph nodes, in female animal no. 64 (LD group) complete dilatation of the uterus, in male animal no. 26 (MD group) enlarged kidneys on both sides and the left kidney was fluid filled as well as dilatation in the left ureters and in female animal no. 81 (HD group) complete dilatation of the uterus.
Macroscopic examination of the decedent animals at necropsy listed in male animal no. 21 (MD group) abnormal dark red colour of the lung, in female animal no. 75 (MD group) abnormal red colour of the lung, in male animal no. 21, no. 24 and no. 25 (MD group) a gas filled stomach, in male animal no. 21 (MD group) a gas filled duodenum and jejunum, in male animal no. 21 and no. 25 (MD group) a gas filled ileum, in male animal no. 21, no. 24 and no. 25 (MD group) a gas filled caecum, in male animal no. 21, and no. 25 (MD group) a gas filled colon, in male animal no. 21 (MD group) a small prostate gland, in male animal no. 24 (MD group) on both sides small seminal vesicles, in male animal no. 24 (MD group) an abnormal spotted coloured and completely enlarged thymus, in male animal no. 40 (HD group) completely enlarged heart, in female animal no. 82 and no. 89 (HD group) abnormal dark red colour of the lung, in male animal no. 36 (HD group) a lung that failed to collapse and was filled with a white fluid, in female animal no. 86 (HD group) the lung failed to collapse, in male animal no. 33 (HD group) the stomach had an abnormal red spotted colour, in male animal no. 31, no. 32, no. 33, no. 36, no. 37, no. 38, no. 40, no. 46 and no. 47 (HD group) and in female animal no. 84, no. 86 and no. 99 (HD group) a gas filled stomach, in male animal no. 31, no. 32, no. 33, no. 36, no. 40 and no. 46 (HD group) and in female animal no. 81 and no. 86 (HD group) a gas filled duodenum, in male animal no. 32, no. 33, no. 36, no. 40 no. 46 and no. 47 (HD group) and in female animal no. 81 and no. 86 (HD group) a gas filled jejunum, in male animal no. 32, no. 36, no. 40 no. 46 and no. 47 (HD group) and in female animal no. 81 and no. 86 (HD group) a gas filled ileum, in male animal no. 31, no. 32, no. 33, no. 36, no. 40, no. 46 and no. 47 (HD group) and in female animal no. 81, no. 85 and no. 86 (HD group) a gas filled caecum, in male animal no. 31, no. 32, no. 33, no. 36, no. 40, no. 46 and no. 47 (HD group) and in female animal no. 81 and no. 86 (HD group) a gas filled colon, in male animal no. 33 (HD group) an abnormal gelatinous content in the urinary bladder, in male animal no. 31, no. 33 and no. 46 (HD group) a small prostate gland and on both sides small seminal vesicles, in female animal no. 85 (HD group) a dilatation in the uterus on both sides, in male animal no. 38 (HD group) and in female animal no. 82 and no. 89 (HD group) a completely enlarged thymus, in female animal no. 84 (HD group) an abnormal red colour of the thymus, in female animal no. 82, no. 84 and no. 89 (HD group) an abnormal spotted colour of the thymus, in male animal no. 85 (HD group) a 1 mm mass in the thymus and in female animal no. 99 (HD group) an enlarged and abnormal spotted coloured thymus with a 1 mm red mass on the right side.
No macroscopic findings were made in recovery animals with exception of an abnormal dark pancreas in the female animal no. 100 (HD group).
The gross findings recorded in the survivors were within the range of normal background changes which may be observed in rats of this strain and age, were incidental gross appearances without corresponding histological changes, or were macroscopic alterations representing normal physiology.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Under the conditions of this study, there were 20 premature deaths in total in the course of the treatment period. In spite of an increased mortality and morbidity in the MD and HD groups, there were no histomorphologic findings that were considered to be due to toxicologically relevant systemic effects exerted by the test item.
In the decedents, necrotizing inflammation was noted in the upper respiratory tract, and the necrotic and inflammatory lesions were found in the lungs and stomach, too. These included:
- Necrotizing inflammation in the larynx, trachea, nasal cavity and maxillary sinus;
- Inflammation at/around the alveolar ducts of the lung, and hypertrophy of the bronchial epithelium;
- Forestomach ulceration, glandular stomach erosion and lymphoid follicle formation in the glandular stomach. In the glandular stomach, mucous cell hypertrophy/hyperplasia was also observed, and this change is known to be induced as a response to irritation.
From these findings, the cause of morbidity in all decedents was the upper respiratory injuries, which were considered to have arisen in relation to the gavage method applied for oral dosing; that is, the regurgitation or gastroesophageal reflux of the test item formulation to the upper respiratory tracts and subsequent aspiration. In addition, the stomach lesions indicate possible irritative physicochemical properties of the test item formulation.
Concurrently, histopathological changes that were considered to be related to toxicologically relevant systemic effects of the test item were not observed in any of the decedents. Thus, in spite the fact that twenty animals in total died or were euthanized prior to the completion of the treatment period, there was no mortality or morbidity caused by toxicologically relevant systemic effects exerted by the test item. Other than the above-mentioned lesions, reactive changes to the inflammatory lesions, stress-related changes, and/or the changes related to a deterioration of animals’ general conditions were observed in decedents. These included: increased granulopoiesis in the bone marrow; diffuse adrenocortical hypertrophy; apoptosis/increased tangible macrophages or lymphoid depletion in the thymus, spleen and lymph nodes; decreased cellularity in the bone marrow; tubular dilatation in the kidney; focal necrosis in the liver; acinar cell hypertrophy in the mandibular glands; reduced secretion in the prostate, coagulating glands and seminal vesicles. However, there were no histologic evidences that were considered to be related to toxicolocally relevent systemic effects of the test item. In the survivors, increased incidences and/or group mean severity grades of infiltration of mixed inflammatory cells and squamous metaplasia of epithelium overlying ventral glands were recorded in the larynx of HD animals sacrificed at the end of their treatment period. In line with the findings in decedents, these were considered to be findings due to the local effects, which arose in relation to the oral gavage method and deemed not to be caused by systemic toxicological effects of the test item. Concurrently, there was no histologic evidence that was considered to be related to any toxicologically relevant systemic effects of the test item.
In the present study, possible toxic effects on fertility were assessed in relation to the reproductive organs. No histomorphological changes of toxicological concern were observed in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones
At the end of the treatment period and after the recovery period, no statistically significant changes were recorded in hormone levels.
Fertility parameters
There were no statistical significances on the testis weight, for mean sperm count and mean sperm motility for all dose groups in the treatment period or after the recovery period. Mean total number of abnormal and normal sperms/findings showed no statistical significances after end of the treatment period or after the recovery period.
No effects on oestrous cycles were reported.
No histomorphological changes of toxicological relevance were observed in testes, epididymides, prostate glands, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse systemic effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a 90-day oral toxicity study according to OECD 408 and in compliance with GLP, no toxicologically relevant systemic effects of the test item were noted in the histopathological examination despite the increased mortality and morbidity in the mid and high dose groups; the NOAEL for systemic toxicity was concluded to be 500 mg/kg bw/day for both sexes under the conditions of this study.