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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:
2016-05-04 to 2016-08-30
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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
September 21, 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate
EC Number:
261-879-6
EC Name:
Bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate
Cas Number:
59719-67-4
Molecular formula:
C24H46N4O6
IUPAC Name:
bis[2-[2-(1-methylethyl)-3-oxazolidinyl]ethyl] hexan-1,2-diylbiscarbamate

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 41 - 46 d
- Weight at study initiation: Males: 158 - 180 g; Females: 105 - 126 g
- Housing: 5 animals of the same sex/ cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
water-free PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of Test Facility and stored at 5 ± 3 °C until use, but not longer than for one day (based on the stability features of the test item in the vehicle).

VEHICLE
- Justification for use and choice of vehicle: The substance rapidly hydrolyses with atmospheric moisture, therefore water free PEG 400 was used as vehicle.
- Concentration in vehicle: 200 mg/mL, 60 mg/mL and 20 mg/mL
- Amount of vehicle: constant treatment volume of 5 mL/kg bw was administered
- Batch no.: 15I040501
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured:
Date of sampling: May 05, 2016; June 13, 2016; July 20, 2016
Date of measurement: May 06, 2016; June 14, 2016; July 21, 2016
The samples were stored in a refrigerator until the analysis.
Measured concentrations varied between 94 and 110 % of the nominal concentrations and all formulations were considered to be homogeneous.
Duration of treatment / exposure:
90 to 91 days (depending on the day of necropsy)
Frequency of treatment:
once daily at a similar time (± 2 hours)
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 200 mg/mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
equivalent to 60 mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
equivalent to 20 mg/mL
No. of animals per sex per dose:
Group 1 and 4: 15 (5 animals of each sex were allocated to the recovery group)
Group 2 and 3: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 1000, 300 and 100 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study with Incozol 4 in the Rat (A 28-Day Repeated Dose Oral Toxicity Study of CG-S4 in Rats; Study no. TBH-1460 (KG-2011-412); GLP) and in agreement with the Sponsor. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once, prior to the first exposure and once weekly thereafter
- Cage side observations included: signs of morbidity and mortality, skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling; special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma (treatment and recovery periods)

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, then weekly during the course of the treatment and recovery periods

FOOD CONSUMPTION
-Time schedule: on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period and prior to test termination
- Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment; at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes, for 16 hours
- How many animals: all animals
- Parameters examined: leukocyte count, erythrocyte count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin concentration, reticulocytes, platelet count, differential white blood cell count, activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment; at the end of the recovery period
- Animals fasted: Yes, for 16 hours
- How many animals: all animals
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, gamma glutamyltransferase activity, alkaline phosphatase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, inorganic phosphate concentration, calcium concentration, sodium concentration, potassium concentration, chloride concentration, albumin concentration, total protein concentration, albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, with all animals
The following organs were examined:
adrenal glands, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), esophagus, eyes (lachrymal gland and Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with cervix and vagina), gross lesions, heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular and mesenteric), muscle (quadriceps), pancreas, pituitary, prostatesalivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder

HISTOPATHOLOGY: Yes (see above), full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) group including recovery groups
Other examinations:
EXAMINATION OF ESTROUS CYCLE
- Time schedule: during the last two weeks of the treatment period (from Day 77/ 78 up to and including Day 90 or 91, depending on the day of the necropsy)

SPERM EXAMINATIONS
Sperm analysis (qualitative and quantative) was made from 10 animals of the control and from 10 animals at high dose group. One-side testes and epididymides were used for enumeration of spermatids and for evaluation of sperm motility and sperm morphology.
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- sperm parameters
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups.
Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse signs of systemic toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily clinical observations. There were no test item related clinical signs during the weekly detailed observations in male or female animals at any dose level during the entire observation period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was slightly reduced at 1000 mg/kg bw/day (male and female) during the treatment period. The slight body weight depression was reversible as there were no significant differences between the control and high dose groups at the end of the post-treatment observation period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was slightly reduced in male and in female animals at 1000 mg/kg bw/day during the treatment period. The slight change recovered up to the end of the post-observation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to their control group, a slight reduction in some red blood cell parameters (red blood cell count, hemoglobin concentration or hematocrit value) was detected in male and female animals at 1000, 300 and 100 mg/kg bw/day at termination of the treatment. The values of RBC, HGB or HCT at 300 or 100 mg/kg bw/day (male or female) remained well within the historical control ranges therefore changes were considered toxicologically not relevant. There were no changes at 1000 mg/kg bw/day in related clinical chemistry parameters or organs at the histopathological examinations referring to deterioration/ destroy or reduced production of red blood cells to substantiate toxicological relevance of these changes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry examinations revealed slight elevation of serum concentrations of calcium and albumin in male animals at 1000 mg/kg bw/day and of serum concentrations of calcium, albumin and total protein in female animals at 1000, 300 and 100 mg/kg bw/day along with elevated urea concentration at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day (female) with respect to the control. There were no histological changes in the kidneys or liver or other signs of renal or hepatic failure or supporting changes in the hematological parameters (erythrocyte count, hematocrit and hemoglobin values) therefore toxicological meaning of these findings are questionable.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observation battery did not demonstrate any treatment-related differences with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 1000, 300 or 100 mg/kg bw/day).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related organ weight changes were noted for the liver in male animals at 1000 mg/kg bw/day and in female animals at 1000 and 300 mg/kg bw/day and for the kidneys in female animals at 1000 and 300 mg/kg bw/day at the termination of the treatment period. The changes in the liver and kidneys weights were fully recovered to control values in male and female animals by the end of post-treatment period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination did not reveal test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period. Histopahtological findings were only observed in single animals equally distributed in the control and high dose groups without showing any dose response relationship.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle
A test item effect on the estrous cycle was not detected at any dose level. There were no significant differences between the control and test item treated groups in the percentage of female animals with irregular or irregular cycles, mean number of cycles or mean cycle length.

Sperm Examinations
The sperm count, morphology and motility of sperm cells were not affected after three months administration of 1000 mg/kg bw/day of the test item. The mean sperm count was similar in animals of the control and 1000 mg/kg bw/day groups at the termination of dosing.
Details on results:
CLINICAL SIGNS
Test item related salivation was observed in male and female animals at 1000 mg/kg bw/day (15/15 male and 13/15 female) with different incidence and duration during the treatment period. Salivation was seen shortly after the daily administration of the test item and it ceased shortly thereafter. It was therefore was judged to be not adverse. Control male and female animals did not salivate therefore salivation was considered to be related to the treatment with the test item.
In one male animal at 1000 mg/kg bw/day, a knot was detected at the inguinal region (right side) as an individual lesion during the last few days of the treatment period (between Days 86 and 90).
The behavior and physical condition of animals in the 300, 100 mg/kg bw/day and in the control group were considered to be normal during the entire observation period.
Soft stool was observed in the bedding material in each cage from Day 4 up to one day after the last treatment. The soft stool (slightly softer than normal) was present in cages of the control and test item treated animals. Therefore it was considered to be not related to the treatment with Incozol 4.
Clinical signs were not detected in animals of control or 1000 mg/kg bw/day during the recovery period. The behavior and physical condition of animals were considered to be normal. Salivation and soft stool ceased one or two days after the last treatment.
The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period (treatment and recovery periods).

MORTALITY
No mortality was observed in any group (control, 1000, 300 or 100 mg/kg bw/day) during the course of study.

BODY WEIGHT
The mean body weight was slightly lower with respect to the relevant control in male animals at 1000 mg/kg bw/day from Day 14 up to the termination of the treatment. The mean body weight gain remained below the control value in male animals at 1000 mg/kg bw/day during the entire study reaching statistical significances on weeks 1, 2, 5, 6, 11 and 13 as well as for the study overall (between Days 0 and 89).
Some statistically significant difference with respect to the control was also detected for the lower mean body weight gain in male animals at 300 mg/kg bw/day between Days 42 and 49 as well as between Days 70 and 77, between Days 84 and 89, and at 100 mg/kg bw/day between Days 0 and 7 and between Days 42 and 49, and between Days 84 and 89. However, these slight changes did not cause relevant or significant changes in the mean body weight of these groups.
The mean body weight was also slightly lower with respect to the relevant control in female animals at 1000 mg/kg bw/day from Day 14 up to the termination of the treatment. The mean body weight gain remained below the control value with statistical significances on weeks 2, 4, 6 and for the study overall in female animals at 1000 mg/kg bw/day. A slightly but statistically significantly lower body weight gain was detected in female animals at 300 mg/kg bw/day when comparing to control between Days 77 and 84.
There were no significant (statistically or biologically) differences between the control and high dose treated male animals during the course of the recovery period.
The mean body weight of high dose female animals was lower than in the control group during the entire recovery period reaching statistical significances on recovery weeks 1, 2 and 3. However the mean body weight gain of these female animals exceed the relevant control value on week 2, 3 and if calculated for the total post-treatment observation period.

FOOD CONSUMPTION
The mean daily food consumption was lower in male animals at 1000 mg/kg bw/day respective to control animals during the entire treatment period with statistical significances on weeks 1, 2, 3, 5 and 12.
Statistically significant differences with respect to the control were observed for the lower mean daily food consumption in female animals at 1000 mg/kg bw/day from week 2 up to the end of the study and at 300 mg/kg bw/day on weeks 9, 11, 12 and13. Similarly, the mean food consumption was slightly but statistically significantly lower than in the control group in female animals at 100 mg/kg bw/day on week 9.
During the course of the recovery period, the mean daily food consumption was comparable in the control and high dose groups for most of the cases. The mean daily food consumption remained below the control value on week 1 of the recovery period in female animals at 1000 mg/kg bw/day, but it was similar to the control during recovery weeks 2, 3 and 4.
Although these differences were of low degree (<20%), regarding the consistent occurrence at 1000 mg/kg bw/day (male and female) a test item influence seemed to be verified. Moreover changes in the mean food consumption correlated with the body weight changes at 1000 mg/kg bw/day (male and female). Slight changes at 300 and 100 mg/kg bw/day were not considered to be toxicologically relevant.

OPHTHALMOLOGICAL FINDINGS
The eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.

ESTROUS CYCLE
There were no significant differences between the control and test item treated groups in the percentage of female animals with irregular or irregular cycles, mean number of cycles or mean cycle length.
Statistical significances with respect to the control were observed in the mean number of days in estrous and diestrous at 1000 mg/kg bw/day and in the mean number of days in pro-estrous at 100 mg/kg bw/day. There were slight differences between the control and 1000 mg/kg bw/day dose in the number of animals in prolonged estrous and in the number of animals in prolonged diestrous. These slight differences between the control and dosed animals were considered to be indicative of the biological variation and not related to the test item. In accordance with this finding, histopathological examinations did not reveal changes in the morphology of uterus or ovaries in this study. Moreover, there was no difference in the estrous cycle parameters (mean number of cycles or mean cycle length).

HEMATOLOGY
Statistically significant changes were revealed in male animals for the lower mean red blood cell count (RBC), hemoglobin concentration (HGB) and hematocrit (HCT), as well as for the lower mean corpuscular hemoglobin content (MCH) and mean corpuscular hemoglobin concentration (MCHC) at 1000 mg/kg bw/day. The mean hemoglobin concentration and hematocrit were also below the relevant control at 300 mg/kg bw/day.
In the female animals, statistically significant changes were revealed for the lower mean percentage of red blood cell count, mean hemoglobin concentration and hematocrit at 1000 and 300 mg/kg bw/day with respect to the control. Additionally, statistical significances with respect to the relevant control were also observed in female animals administered with 1000 mg/kg bw/day for the lower mean percentage of the eosinophil granulocytes (EOS), lower mean corpuscular volume (MCV), lower mean corpuscular hemoglobin content and hemoglobin concentration and mean percentage of reticulocytes (RET). At 300 mg/kg bw/day females, the mean percentage of basophil granulocytes (BASO) and mean corpuscular hemoglobin content were below the control value while the mean activated partial prothrombin time (APTT) was slightly elevated. In female animals at 100 mg/kg bw/day, the mean percentage of basophil granulocytes and mean hemoglobin concentration were slightly below the appropriate control value.
All examined hematological parameters were comparable in male animals in the control and high dose group at the end of the recovery period.
In the female animals of recovery group at 1000 mg/kg bw/day, a statistically significant difference with respect to the relevant control was observed for the lower mean corpuscular hemoglobin content.
Changes in other hematological parameters were judged to be toxicologically not relevant as values were of low degree and remained well within the historical control ranges (EOS, BASO, MCV, MCH, MCHC, RET, APTT). Moreover, some of these changes were only present at the lower dose groups but not at the high dose group (BASO, APTT) therefore its biological or toxicological importance is equivocal.

CLINICAL CHEMISTRY
In the male animals, statistically significant differences were observed in the enzyme activities of alanine aminotransferase (ALT; reduced), aspartate aminotransferase (AST; elevated) and alkaline phosphatase (ALP; elevated) at 1000 mg/kg bw/day. Similarly, statistical significances were revealed with respect to the relevant control for higher mean concentrations of urea, calcium (Ca2+), sodium (Na+), potassium (K+) and (albumin) and for the lower mean concentration of inorganic phosphorous (Pi) in male animals administered with 1000 mg/kg bw/day. The mean activity of alkaline phosphatase was higher than in the control group in male animals at 300 mg/kg bw/day. The mean inorganic phosphorous concentration was also below the control value in male animals at 300 and 100 mg/kg bw/day while the mean calcium concentration exceeded that of the controls in male animals at 300 mg/kg bw/day at the end of the treatment period.
In the female animals, statistical significances were revealed with respect to the appropriate control for the lower mean activity of alanine aminotransferase and for the higher mean concentrations of calcium, albumin and total protein (TPROT) at 1000, 300 and 100 mg/kg bw/day. The mean urea concentration was also elevated in female animals at 1000 and 300 mg/kg bw/day at the end of the treatment period.
At the end of the recovery period, some sporadic statistically significant differences with respect to the control were observed for examined clinical chemistry parameters in the high dose treated group: the lower mean concentration of creatinine (CREA) in male animals and the higher mean concentrations of calcium, albumin and total protein in female animals.
Changes in the alanine aminotransferase activity in male and female animals and in inorganic phosphorous in male animals were considered toxicologically not relevant because there were no supporting histopathological lesions in the liver or bone. The sporadic statistical differences in male animals (AST, ALP, Na+, K+) at the end of the treatment period and in male or female animals (CREA, or Ca2+, ALB, TPROT, respectively) at the end of the recovery period were considered to be of little or no biological relevance. The mentioned differences between the control and test item treated groups were statistically significant but were small and all values remained within the historical control ranges for these parameters. Therefore, these findings were finally considered to be not of toxicological relevance.

FOB
The behavior and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

ORGAN WEIGHTS
Statistical significant differences with respect to the control were revealed for the lower mean fasted body weight, the higher mean liver weight and lower mean thymus and epididymides weights of male animals at 1000 mg/kg bw/day at the termination of the treatment. The mean kidney weight was also higher than in the control group at 100 mg/kg bw/day male animals. Statistical significances were also observed in higher mean organ weights relative to body weight in case of the liver and kidney in male animals at 1000 and 300 mg/kg bw/day and in kidneys of male animals at 100 mg/kg bw/day. The mean liver and kidney weights both relative to brain weight exceeded the control in male animals at 1000 and 100 mg/kg bw/day, respectively. The mean heart weight relative to brain weight was statistically significantly lower than in the control in male animals at 1000 and 300 mg/kg bw/day. The mean thymus and epididymides weights of male animals at 1000 mg/kg bw/day were also lower than in the control group at the termination of the treatment.
In the female animals, statistical significances were observed for the lower mean fasted body weight at 1000 mg/kg bw/day and the higher mean liver and kidney weights ( absolute and relative to body and brain weights) at 1000 and 300 mg/kg bw/day at the end of the treatment period. The mean brain weight relative to body weight exceeded the control value while the mean body weight relative to brain weight was below the control value in female animals at 1000 mg/kg bw/day.
At the end of the recovery period, statistical significances were revealed for the higher mean kidney weight (absolute and relative to body and brain weight) and mean liver weight relative to body weight as well as for the lower mean testes weight in male animals at 1000 mg/kg bw/day with respect to the control. In female recovery animals of 1000 mg/kg bw/day, statistically significant difference with respect to the control group was seen for the higher mean liver and kidney weight both relative to body weight.

SPERM EXAMINATIONS
The percentage of motile cells and cells with normal morphology were slightly higher in the high dose (1000 mg/kg bw/day) treated animals with respect to the control. The percentage of immotile sperms and sperms with not normal morphology (separated head and tail) was slightly lower in animals at 1000 mg/kg bw/day with respect to the control groups.

GROSS PATHOLOGICAL FINDINGS
Individual macroscopic changes were noted for some male animals in the control and high dose groups: renal pyelectasia (1/10 control), pale and nutmeg-like patterned liver (1/10 at 1000 mg/kg bw/day) and knot in the inguinal region (1/10 at 1000 mg/kg bw/day) were detected. These findings are common in untreated experimental rats.
In the female animals, slight or moderate hydrometra in the uterus was observed as follows: 2/10 control, 3/10 at 100 mg/kg bw/day, 3/10 at 1000 mg/kg bw/day. Hydrometra is indicative of female sexual cycle. There were no pathological alterations in the uterus at the histopathological examination therefore it was considered toxicologically not relevant.
There were no macroscopic changes in the male animals observed for four weeks post-treatment (control and 1000 mg/kg bw/day) at the necropsy. In some female animals hydrometra was observed at the end of the recovery period (1/5 control and 2/5 at 1000 mg/kg bw/day).

HISTOPATHOLOGY
Pulmonary alveolar emphysema was observed in some animal (2/10 male control, 1/10 female at 1000 mg/kg bw/day) in connection with the hypoxia, dyspnea and circulatory disturbance, developed during the exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) occurred in the lungs (2/10 male and 1/10 female control; and 1/10 male and 2/10 female at 1000 mg/kg bw/day) and it was considered as a physiological phenomenon as this finding showed a higher incidence in male control animals than in the high dose group.
Alveolar histiocytosis was found in some control and treated animals (3/10 male and 4/10 female control; 3/10 male and 1/10 female at 1000 mg/kg bw/day). Alveolar histiocytosis is a common incidental finding in elder rats and consists of small collections of alveolar macrophages with abundant foamy (phospholipid-containing) cytoplasm. These cells are often sub-pleural or located in the more peripheral regions of the lung.
Individual diseases were observed in single animals of the control and 1000 mg/kg bw/day groups. One side pyelectasia was seen in the kidney (without other pathological lesion – degeneration, inflammation, fibrosis etc.) in one male control animal (1/10). Minimal centrilobular vacuolar degeneration was present in the liver in one male animal (1/10) at 1000 mg/kg w/day. Cystadenoma was observed in the skin in another male animal (1/10) at 1000 mg/kg w/day.
The dilatation of the uterine horns was found in some female animals (2/10 control; 2/10 at 1000 mg/kg bw/day). Hydrometra is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrous phase – of the inner genital organs.
At the end of the recovery period, pulmonary changes were also present in some animals: alveolar emphysema in 1/5 control male; hyperplasia of bronchus associated lymphoid tissue in 1/5 control male.
Dilatation of uterine horns was also detected in female animals at the end of the recovery period: 1/5 control and 2/5 at 1000 mg/kg bw/day.
Morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the alimentary system, the pancreas, the cardiovascular system, immune system, the hemopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central and peripheral nervous system was not detected.
The structure and the cell morphology of the endocrine glands was the same in the control and high dose treated animals.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The results of the 90-Day Repeated Dose Oral Toxicity Study of Incozol 4 in Rats showed slight reduction in some red blood cell parameters in male and female animals in a dose related manner at 300 and 1000 mg/kg bw/d. The haematological parameters remained well within the historical control ranges at 300 mg/kg bw/d. Thus, the no-observed-adverse-effect-level (NOAEL) of Incozol 4 was set to 300 mg/kg bw/day under the conditions of this study.
Executive summary:

The subchronic oral toxicity of the test substance was assessed in a 90 day study according to OECD guideline 408 and EU method B.26.

The test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20 mg/mL, 60 mg/mL and 200 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals/sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89. Then they were observed without administration for four weeks (recovery observations).

Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. The estrous cycle of each female animal was examined for two weeks before necropsy. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups.

The results of the study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. Slight salivation was observed in most of the animals at 1000 mg/kg bw/day (15/15 male and 13/15 female) with different incidence and duration during the treatment period. Animals salivated shortly after the daily administration of the test item and it ceased shortly thereafter and was therefore judged to be not adverse. The body weight development was slightly reduced at 1000 mg/kg bw/day (male and female) during the treatment period. The slight body weight depression was reversible as there were no significant differences between the control and high dose groups in the mean body weight at the end of the post-treatment observation period. The mean daily food consumption was slightly reduced in male and in female animals at 1000 mg/kg bw/day during the treatment period. The slight change recovered up to the end of the post-observation period. There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (1000, 300 and 100 mg/kg bw/day). A slight reduction in some red blood cell parameters (red blood cell count, hemoglobin concentration or hematocrit value) was detected in dose related manner in male and female animals at 1000 and 300 mg/kg bw/day with respect to the relevant control at termination of the treatment. The values of RBC, HGB or HCT at 300 mg/kg bw/day (male or female) remained well within the historical control ranges therefore changes were considered toxicologically not relevant. Moreover, there were no related changes in clinical chemistry parameters or organs at the histopathological examinations referring to deterioration/ destroy or reduced production of red blood cells to substantiate toxicological relevance of these changes at 1000 mg/kg bw/day. Clinical chemistry examinations revealed slight elevation of serum concentrations of calcium and albumin in male animals at 1000 mg/kg bw/day.Serum concentrations of calcium, albumin and total protein were also elevated in female animals in a dose related manner at 1000, 300 and 100 mg/kg bw/day. Furthermore an elevated urea concentration at 1000 mg/kg bw/day (male and female) and at 300 mg/kg bw/day (female) was observed with respect to the control. There were no related changes in the hematological parameters or structural (histopathological) alterations in relevant organs. Therefore these findings were considered to be signs of adaptation of the organs (liver, kidneys) to the altered demands. Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period. Test item related organ weight changes were noted for the liver in male animals at 1000 mg/kg bw/day and in female animals at 1000 and 300 mg/kg bw/day as well as for the kidneys in female animals at 1000 and 300 mg/kg bw/day at the termination of the treatment period (organ weights were elevated. However there were no related histopathological alterations in the liver or kidney tissue. Therefore these changes were considered as signs of adaptation. The changes in the liver and kidney weights were fully recovered to control values in male and female animals by the end of post-treatment period. Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 1000 mg/kg bw/day. Histological examination did not reveal test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.

Under the conditions of the present study, 1000 mg/kg bw/day dose of Incozol 4 induced salivation (male and female), changes in body weight development (male and female), and food consumption (male and female) after consecutive 90-day oral (gavage) administration to Hsd.Han:Wistar rats. These changes were fully reversible in male and female animals up to the end of the four weeks post-treatment period. Changes in some red blood cell parameters (red blood cell count, hemoglobin concentration or hematocrit; male or female), in biochemical parameters (calcium, albumin or total protein and urea; male or female) or organ weights (liver or kidneys; male or female) might be indicative of a possible test item influence however without any structural (histopathological) alterations to substantiate their toxicological relevance. The slight changes in some of the above mentioned parameters (hematology, clinical chemistry, organ weights) at 300 or 100 mg/kg bw/day remained well within or were marginal to the range of historical control and without supporting changes in related parameters therefore were judged to be toxicologically not significant.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 300 mg/kg bw/d.