Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-12 - 2012-08-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles of Good Laboratory Practice as revised in 1997 (ENV /MC/CHEM(98) 17) & with the revised German Principles of Good Laboratory Practice according to Annex I German Chemicals Act (Bundesgesetzblatt, Volume 2008, Part I, No 28, 1173-1184, 2008
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Bis-(2,6-diisopropylphenyl )carbodiimid
- Chemical name: N,N-bis[2,6-di(propan-2-yl)phenyl]methanediimine
- Substance type: organic
- Physical state: waxy solid, slightly yellow if molten
- Analytical purity: 99.9 %
- Expiration date of the lot/batch: 17-MAR-2013
- Storage condition of test material: Room temperature, light protection

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - Wistar rats (Hsd Cpb:WU)
- Source: Harlan Laboratories BV, Kreuzelweg 53, 5961 Horst, The Netherlands
- Delivery of animals: 2011-08-11
- Age at study initiation: age at arrival at the testing facility: 6-7 weeks
- Weight at study initiation: mean Initial Weights at Study Start: males: 217.8 g, females: 170.9 g
- Housing: From arrival to start of study individually in Makrolon cages Type IIa. From tattooing to necropsy in groups with three (first cage) or two rats in Makrolon cages Type IV. Bedding material: Low-dust wood granules (Lignocel BK 8-15; supplier: Ssniff Spezialdiäten Inc. Soest/Westfalen, Germany; manufacturer: J. Rettenmeier, Ellwangen-Holzmühle, Germany). Wooden blocks for environmental enrichment were added to each cage. As soon as necessary, they were replaced by new ones. Supplier: Tapvei OY, 73620 Kortteinen, Finland.
- Diet (e.g. ad libitum): Ad libitum in cage lids: Provimi Kliba Maus/Ratte-Haltung-GLP, Article No.: 3883.PM S15 (pellet) by Provimi Kliba SA, 4303-Kaiseraugst, Switzerland;
- Water (e.g. ad libitum): Tap water ad libitum in polycarbonate bottles; Specification of contaminants: According to actual German drinking water standards.
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2°C, temperature was recorded continuously
- Humidity (%): Approximately 55 %., relative humidity was recorded continuously
- Air changes (per hr): ≥ 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

OTHER:
- Animal identification: tail tattoos and coloured cage cards

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item (test substance) was solved in the vehicle and administered by gavage. The administration volume was 5 mL/kg body weight per day. The formulations were prepared as needed taking into account the analytically determined stability. For the preparation of the formulations, a test item content of 100% was assumed for calculation. Administration Volume: 5 mL/kg b.w. (volume was based on body weights)

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle has been proven to be a suitable vehicle for lipid soluble substances and has been used frequently in subacute and chronic studies
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment the stability of the formulations was analytically confirmed. For these analyses dosage forms were prepared according to the procedure intended for the dosage forms in the study. Analyses were carried out under study No. F1012107. The dosage forms prepared for stability analysis were analysed shortly after preparation and 3, 4, 7, 8 and 15 days thereafter. The analysis revealed that the test item was stable over 7 days within the defined limits. Content checks of formulations (including controls) given to the animals were determined two times during the study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily for at least 28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
4 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
16 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected according to results obtained in a previous oral 2-weeks pilot toxicity study (No. T0083108) performed in rats at Bayer Pharma AG, where 0, 4, 8, 16 or 24 mg/kg Bis-(2,6-diisopropylphenyl) carbodiimide were administered in corn oil to 3 male rats. The administration volume was 5 mL/kg.
In-life data and organ weight measurements showed following treatment-related changes: One animal of the highest dose group was killed in moribund condition on day 8, showing breathing difficulties and poor general condition. At necropsy the abdomen was filled with fluid. Body weight gain was reduced at the doses of 16 mg/kg (10%) and 24 mg/kg (20%). Food intake was reduced dose-dependently starting at the dose of 16 mg/kg (≥ 15%) and water intake was reduced dose-dependently starting at the dose of 4 mg/kg (≥16%). In addition, absolute organ weights of kidneys, spleen and thymus were reduced at 16 mg/kg due to the decrease in body weights. Absolute and relative weights of adrenals were increased dose-dependently starting at all doses: 10% (4 mg/kg), 18% (8 mg/kg), 25% (16 mg/kg) and 60% (24 mg/kg). The testes weights were reduced starting at 16 mg/kg.
In addition, data of a 4-week subacute oral toxicity study with 5 administrations per week in rats were available (Bayer, Lorke, 1962), where doses of ≥16 mg/kg exhibited clear-cut toxicity and a dose of 8 mg/kg was tolerated without symptoms.
Based on all these data, the dose of 16 mg/kg was considered to be the MTD for a 28 day study, mainly because of the reduced body weight gain in the pilot study and was thus chosen as the high dose. The low dose of 1 mg/kg was expected to be tolerated without any adverse effects, as 4 mg/kg caused only very minor effects. The mid dose of 4 mg/kg represents the geometric mean between low and high dose and is thus well suited for evaluation of dose-response-relationship.

- Rationale for animal assignment (if not random): At the start of the study male and female animals were assigned to the dose groups using the PTS random program

Examinations

Observations and examinations performed and frequency:
Inspection of Animals for Morbidity and Mortality: twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Time point: daily about 30 minutes after administrtion
- evaluated parameters: posture, piloerection, gait abnormalities, involuntary motor movements, vocalization, others.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (including open Field Observation (OFO))
- Once before the start of treatment and once weekly thereafter animals were clinically examined in detail including observations in a standard arena (open field) for behavioural observations. Any clinical signs (findings) and abnormalities were recorded. Body surfaces and orifices, posture, general behaviour, breathing and excretory products were assessed. Findings and abnormalities were recorded either using a coding system or uncoded.
- evaluated parameters during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation, nasal discharge, salivation, stains, others.
- evaluated parameters during open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behaviour, gait abnormalities, vocalization, arousal, rearing, defecation, urination.

BODY WEIGHT: Yes
- Time schedule for examinations: daily
- Body weights of animals were determined before the study start and daily thereafter up to scheduled necropsy.
- Body weights were used to calculate the appropriate administration volumes.
- The corresponding administration volumes, which were recorded on-line, were filed together with the study raw data.
- Furthermore, body weights were also recorded immediately before scheduled necropsies for calculation of relative organ weights.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)
- Food and water intake was determined per cage at comparable periodical intervals (e.g. weekly). These primary data were then used to calculate the group means for each period of approximately 7 days. The weight of the food/water offered at the start of the measurement period minus the food/water at the end of the period is defined as the food/water consumption of the animal in g.
- On the basis of these data the following parameters were calculated: -for each interval: daily food intake per animal, mean daily food intake per animal, mean daily food intake per kg body weight; - for the total period: measurement of mean food intake per animal and day, mean food intake
per kg body weight and day; - cumulative food intake per animal and cumulative food intake per kg body weight.
- Comparable calculations were done for water intake.

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly
- - Food and water intake was determined per cage at comparable periodical intervals (e.g. weekly). These primary data were then used to calculate the group means for each period of approximately 7 days. The weight of the food/water offered at the start of the measurement period minus the food/water at the end of the period is defined as the food/water consumption of the animal in g.
- On the basis of these data the following parameters were calculated: -for each interval: daily food intake per animal, mean daily food intake per animal, mean daily food intake per kg body weight; - for the total period: measurement of mean food intake per animal and day, mean food intake
per kg body weight and day; - cumulative food intake per animal and cumulative food intake per kg body weight.
- Comparable calculations were done for water intake.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 24
- Anaesthetic used for blood collection: Yes (CO2) and No - see below
- Animals fasted: No
- How many animals: all
- Clinical pathology of blood samples was performed on all animals alive near termination. In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
- The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasting, non-anaesthetized animals.
- The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasting animals anaesthetized with CO2/air.
The blood obtained was treated as follows: The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant). The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate. The samples for the determinations of electrolyte concentrations were collected in tubes with z-gel. The samples for other biochemical tests were heparinized.
- The blood samples for glucose determinations were mixed with perchloric acid ( 1 + 1 0) to precipitate proteins.
- The following haematological parameters were determined in peripheral blood: Differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin concentration, haematocrit, leukocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 24
- Animals fasted: No
- How many animals: all
- Clinical pathology of blood samples was performed on all animals alive near termination. In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
- The blood samples for determination of glucose concentrations were taken from the caudal vein of non-fasting, non-anesthetized animals.
- The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasting animals anesthetized with CO2/air.
- The following parameters were determined: Alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, total bilirubin, cholesterol, creatinine, total protein, urea, glucose, potassium, sodium, gall acids.
- After determination of parameters mentioned above, remaining plasma and serum was frozen and stored at -80°C; additionally T3, T4 and TSH were determined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
- parameters tested: approach response, touch response, auditory response, tail pinch response, pupil size, pupil response, righting reflex, body temperature, grip strength, foots play.
- parameters tested during open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behaviour, gait abnormalities, vocalization, arousal, rearing, defecation, urination.
- parameters tested concerning motor activity: Motor activity of male and female rats were measured on days 22 and 23 (absolute), respectively. Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session and activity during each 10-minute interval. Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

OTHER:
The purpose of the FOB is to use noninvasive procedures to detect gross functional effects in rats resulting from chemical exposure and to quantify behavioral and neurologic effects. The functional observational battery includes home cage and open field observations, neuromuscular and sensorimotor tests involving handling of the rat. Scoring criteria and explicitly defined scales were used to rank the severity of observations that cannot readily be quantified. On the day of observation the appropriate animals were placed in the correct sequence that
had been established for testing on that day. If possible this placement was done by someone who was not associated with performing observations and measurements on that day. Observations for all animals were performed by the same observer during the study, with a second person recording the measurements. Functional observations were performed once (not blind) on day 21 and 22 (relative), which included home cage observation, observation during handling, behaviour in an open field, reflex/physiological observations and measurement of grip strength.

The figure-eight maze is an established and widely-used automated activity measuring device that can be used to detect both increases and decreases in activity. Animals received the test item some time before the performance of the MA. Each maze consists of a series of inter-connected alleys converging on a central arena and covered by transparent acrylic plastic. Each maze has eight infrared emitter/detector pairs to measure activity. Each time a beam is interrupted, an activity count is registered. Animals were tested individually for 60 minutes in one of eight figure-eight mazes. Motor activity was measured as the number of beam interruptions that occur during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Motor/Locomotor Activity: day absolute 22, 23
Sacrifice and pathology:
Clinical Pathology:
Haematology: day 24
Clinical Chemistry: day 24
Functional Observation Battery: day relative 21, 22
Motor/Locomotor Activity: day absolute 22, 23
Necropsy: day absolute 29/30

GROSS PATHOLOGY: Yes
All animals living on the date of their scheduled necropsy and all animals to be killed in moribund state were sacrificed by exsanguination under isoflurane anaesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination.
Changes were described in terms of localization, size, colour and consistency whenever appropriate.
Animals, which died spontaneously during the study, were necropsied at the earliest opportunity. From these animals the organs and tissues were handled as described above.
The following organs of the animals killed at the end of the treatment were weighed before fixation: Brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides (both), testes (both), ovaries/oviducts (both) and uterus/cervix.
The organ weights are specified in both absolute and relative terms. The relative weights were calculated in % of the terminal body weight.

HISTOPATHOLOGY: Yes
The fixed material was retained.
Statistics:
Statistical tests on body weights and weight gain as well as on absolute organ weights were done using the Dunnett Exact Homogeneous Test. For relative organ weights the Dunnett Exact Homogeneous Test after log. Transformation was used.
If primary food and water intake data were recorded, the calculated food/water intake per animal was evaluated using adjusted Mann-Whitney U-tests.
The Dunnett Exact Homogeneous or Heterogeneous Test, the Dunnett Exact Homogeneous Test after log. Transformation or the Bonferroni/Mann-Whitney U-test was used for clinical pathology parameters.
Descriptive statistics were provided per sex, dose group and time point for all parameters that were recorded with a specified unit. This included measures of general tendency (mean and median (median not given for food and water intake)) and general variability (standard deviation, minimum and maximum) as appropriate.
For continuous variables, the statistical test procedure was based on prior knowledge of the respective variable derived from previous studies. For normally distributed variables with equal variances across treatment groups Dunnett's tests were performed. Heteroscedastic normally distributed variables were analyzed using appropriately adjusted Dunnett's tests, using Satterthwaite adjustments for the degrees of freedom and taking the different variances within the groups into account. For log-normally distributed variables, Dunnett's tests were performed after log transformation of the original values. If experience with historical data indicated that the assumptions for parametric analyses are violated, Bonferroni-adjusted Mann-Whitney U-tests were employed in the analyses. For small sample sizes, the exact version of this test was used.
Statistical tests were not performed for groups, which were smaller than 3.
All statistical tests were performed using standard procedures within the PATH/TOX SYSTEM.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Survival of the animals was not affected in males (all dose groups) and in females (low and mid dose group). Two high dose females (No. 37 and 40) were sacrificed in moribund state on days 11 and 16 and one high dose female (No. 36) was found dead on day 29. All these animals had clinical symptoms prior to death/sacrifice. Clinical observations revealed no toxicological relevant findings in males (all dose groups) and in females (low and mid dose group). In high dose group, 4 of the 5 female animals showed clinical symptoms including piloerection and paleness starting in week 2. In addition, curved back was found towards the end of the treatment period. The animals which died prematurely or were found dead also showed decreased motility, high-stepping gait, sunken flunks, accelerated breathing, and narrowed eyelids on the days prior to death or sacrifice.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of the animals was not affected in males (all dose groups) and in females (low and mid dose group). Two high dose females (No. 37 and 40) were sacrificed in moribund state on days 11 and 16 and one high dose female (No. 36) was found dead on day 29. All these animals had clinical symptoms prior to death/sacrifice. Clinical observations revealed no toxicological relevant findings in males (all dose groups) and in females (low and mid dose group). In high dose group, 4 of the 5 female animals showed clinical symptoms including piloerection and paleness starting in week 2. In addition, curved back was found towards the end of the treatment period. The animals which died prematurely or were found dead also showed decreased motility, high-stepping gait, sunken flunks, accelerated breathing, and narrowed eyelids on the days prior to death or sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were clearly reduced in male & female animals in the high dose group. In males, body weight was 7.8% lower than in controls with cumulative body weight gain being decreased by 28.7%. In females, the effect was more pronounced.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake in the low and mid dose groups was comparable to controls. At the high dose, a decrease in food intake was found in both sexes with the effect being more pronounced in female animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, thrombocyte (THRO) and neutrophilic granulocyte count (NEUTRO) were reduced in females and lymphocyte count (LYM) and eosinophilic granulocyte count (EOS) were reduced in both sexes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, a slight increase in alanine aminotransferase was found in males and a slight increase in higher alkaline phosphatase was found in both sexes. In addition, there was an increase in glucose and cholesterol in both sexes.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the high dose, absolute and relative weights of ovaries and uterus were reduced in females. In addition, absolute and relative kidney weight was lower in male animals.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In one high dose male, the rare finding of pancreatic acinar cell carcinoma was observed. In the absence of any other proliferative finding of the exocrine pancreas in this study, a spontaneous origin is most likely.
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality
Survival of the animals was not affected in males (all dose groups) and in females (low and mid dose group). Two high dose females (No. 37 and 40) were sacrificed in moribund state on days 11 and 16 and one high dose female (No. 36) was found dead on day 29. All these animals had clinical symptoms prior to death/sacrifice.
Clinical Observations
Clinical observations revealed no toxicological relevant findings in males (all dose groups) and in females (low and mid dose group).
In high dose group, 4 of the 5 female animals showed clinical symptoms including piloerection and paleness starting in week 2. In addition, curved back was found towards the end of the treatment period. The animals which died prematurely or were found dead also showed decreased motility, high-stepping gait, sunken flunks, accelerated breathing, and narrowed eyelids on the days prior to death or sacrifice.

BODY WEIGHT AND WEIGHT GAIN
Body weights were clearly reduced in male & female animals in the high dose group. In males, body weight was 7.8% lower than in controls with cumulative body weight gain being decreased by 28.7%. In females, the effect was more pronounced with body weight 16.8% lower than in controls and the cumulative body weight gain being reduced by 84.4% (see Table 1).
In female animals(low and mid dose group), body weight development was comparable to controls within the first 3 weeks of treatment. In the last week, a slightly lower body weight gain was found in these animals, so that the cumulative body weight gain was slightly lower than in controls (-3.4% and -8.2%). The absolute body weights in these groups were 2.0 and 2.2% lower than in the control. When looking at the individual values, no trend was observed, so that these differences are considered to be within normal variability in rats undergoing procedures. The extent of change is too low to be of biological relevance, so that this difference is not considered as indicative of an adverse effect.

FOOD CONSUMPTION
Food intake in the low and mid dose groups was comparable to controls. At the high dose, a decrease in food intake was found in both sexes with the effect being more pronounced in female animals.

WATER CONSUMPTION
The water intake of males was not altered in any dose group. In females, water intake was decreased at the high dose. This finding is considered a secondary finding due to lower food intake.

HAEMATOLOGY
No relevant changes in haematology were found in the low and mid dose groups. In the high dose group, thrombocyte (THRO) and neutrophilic granulocyte count (NEUTRO) were reduced in females and lymphocyte count (LYM) and eosinophilic granulocyte count (EOS) were reduced in both sexes.
The lower eosinophilic granulocyte count found in both sexes of the mid dose group was not considered to be biologically relevant, as all individual values were well within the range of reference values (mean ± 2 standard deviations).
The prolonged Hepato Quick (Hep-Quick) found in high dose females was not considered to be toxicologically relevant, as the values of the individual animals were within the range of reference values (mean ± 3 standard deviations) and since even in the control group, there was a trend towards values within the upper range of reference values.
The lower MCHC in males in the mid dose group is considered a chance finding because of the lack of dose dependence.

CLINICAL CHEMISTRY
Clinical chemistry did not reveal any relevant findings in the low and mid dose group.
In the high dose group, a slight increase in alanine aminotransferase (ALAT) was found in males and a slight increase in higher alkaline phosphatase (APh) was found in both sexes. In addition, there was an increase in glucose and cholesterol (CHOL) in both sexes and an increase in urea in female animals. Protein and albumin was slightly decreased in females.
Thyroid hormones were in the normal range at all dose levels.
All findings listed in the following were considered without toxicological relevance:
The higher values in alanine aminotransferase (ALAT) found in males (mid dose group) is not considered biologically relevant, as all individual values were within the range of reference data (mean ± 2 standard deviations) and as there was no obvious trend compared to the other groups. The decrease in aspartate aminotransferase (ASAT) in female animals is without toxicological relevance, as only an increase is indicative of any organ damage. In addition, the higher ASAT values found in single female animals at the low and mid dose are considered isolated chance findings, since there was no trend and no relation to the administered dose level.
The lower creatinine (CREA) in females at the high dose, and the lower total bilirubin (Bili-t) in males in all dose groups were not considered to be of any biological relevance, as all values were in the range of historical control data (mean ± 2 standard deviations). The values observed in gall acids (S-Bile) in males were in the normal range, the difference to control was due to the high value in a single control male. The higher values in gall acids in the females (mid dose group) are of no toxicological relevance, because there was no trend and no dose-dependency.
In addition, in mid dosed males, potassium (K) values were higher. All individual values of the mid and high dose groups were in the range of historical control data and there was not trend and no dose-dependency, so that this finding is of any toxicological relevance.

NEUROBEHAVIOUR
Motor Activity Assessment
The activity determination over the entire 60-minute observation period revealed no statistically significant effect on motor (MA) and locomotor activity (LMA) up to the highest concentration tested..
Mean and individual motor (MA) and locomotor (LMA) activity data were determined for both the entire session (60-minute) MA and LMA and also the 10-minute intervals.

ORGAN WEIGHTS
There was no effect on organ weights in the low and mid dose groups.
At the high dose, absolute and relative weights of ovaries and uterus were reduced in females. In addition, absolute and relative kidney weight was lower in male animals.
The higher absolute weight of adrenal s in low dose males as well as the increase in relative thymus weight in mid dose females are considered chance findings because of the lack of dose dependency.

GROSS PATHOLOGY
No test item related findings occurred at necropsy and histpathological evaluation in the low and mid dose groups.
Intercurrent mortality was observed in high dose females (3/5 animals) due to myocardial degeneration/myocarditis associated with atrial thrombosis in 2 of the decedents. Gross pleural effusion and mesenteric oedema in these animals are likely related to cardiac changes. Slight myocarditis was also seen in one high dose female and sacrificed as scheduled.
A variety of other findings was encountered in the high dose groups:
- in the liver bile duct hyperplasia and hepatocellular hypertrophy;
- in the lymphoid organs depletion which is often seen in conjunction with deteriorated general condition;
- in the mesenteric lymph nodes increased non-lymphocytic cellularity in medullary cords;
- in the femoral bone increased metaphyseal spongiosa at the diaphysis;
- in the kidneys nephropathy with glomerular and tubular alterations;
- in the pancreas increased apoptosis, mitosis and depletion of zymogen granules of acinar cells;
- interstitial oedema as well as perivascular inflammatory infiltration of mesenteric fat tissue;
- in the thyroid gland flattened follicular epithelia;
- in the female genital tract a marked increase in the amount of large active corpora lutea in the ovaries, atrophy of ovarian follicles, atrophy of oviducts, uterus and vagina.
In one high dose male, a pancreatic acinar cell carcinoma was observed, which is a rare finding. In the absence of any other proliferative finding of the exocrine pancreas in this study and also considering the short duration of treatment, a spontaneous origin is most likely.

OTHER FINDINGS
Functional Observational Battery
Functional observations revealed no treatment-related effects up to the highest concentrations tested.

Effect levels

Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw (total dose)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; haematology; clinical chemistry; gross pathology; organ weights

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study was performed according to OECD TG407 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. The test material did induce mortality and treatment-related clinical signs in the high dose group. A NOAEL was identified (4 mg/kg bw).
Executive summary:

Bis- (2,6-diisopropylphenyl )carbodiimid was administered in a study according to OECD 407 by gavage to 5 male and 5 female Wistar (Hsd Cpb: WU) rats per dose group in daily doses of 0, 1, 4 or 16 mg/kg body weight for a period of at least 28 days (Popp, 2012). As vehicle cornoil was used. The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical pathology of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation. Functional observational battery (FOB) and motor and locomotor activity measurements (MA/LMA) were done. Stability test revealed the test substance formulations to be stable during the period use. Content checks during the study revealed correct concentrations of the test substance in the formulations used.

All male animals survived until scheduled necropsy. In females, survival was not affected up to 4 mg/kg. At the high dose of 16 mg/kg, 2 females were sacrificed in moribund state and one 16 mg/kg female died unscheduled showing distinct clinical symptoms. The male animals were not affected. Clinical observations revealed no relevant findings in males up to 16 mg/kg and in females up to 4 mg/kg. At the dose of 16 mg/kg, 4 of the 5 female animals showed clinical symptoms including piloerection and paleness starting in week 2. In addition, curved back was found towards the end of the treatment period. The animals which died prematurely or were found dead also showed decreased motility, high-stepping gait, sunken flunks, accelerated breathing, and narrowed eyelids on the days prior to death or sacrifice. No relevant changes in body weight were found at 1 and 4 mg/kg. Body weight was decreased in males and females dosed at 16 mg/kg (-7.8% and -16.8% compared to control) with body weight gain being moderately to severely reduced (-28.7% and -84.4%). Food intake was decreased at 16 mg/kg in both sexes. Water intake decreased at 16 mg/kg in females only. No relevant changes in hematology were found at the doses of 1 and 4 mg/kg. At the high dose of 16 mg/kg, thrombocyte and neutrophilic granulocyte count were reduced in females and lymphocyte count and eosinophilic granulocyte count were reduced in both sexes. Clinical chemistry did not reveal any relevant findings at the doses of 1 and 4 mg/kg. At the high dose of 16 mg/kg, a slight increase in alanine aminotransferase was found in males and a slight increase in higher alkaline phosphatase was found in both sexes. In addition, there was an increase in glucose and cholesterol in both sexes and an increase in urea in female animals. Protein and albumin was slightly decreased in females. Thyroid hormones were in the normal range at all dose levels. Organ weights were not altered at 1 and 4 mg/kg. At the high dose of 16 mg/kg, absolute and relative weights of ovaries and uterus were reduced in females. In addition, absolute and relative kidney weight was lower in male animals. Necropsy and histopathological evaluation revealed no alterations at 1 and 4 mg/kg. At 16 mg/kg, the intercurrent mortality in females (3/5 animals) was due to myocardial degeneration/myocarditis associated with atrial thrombosis in 2 of the decedents. Gross pleural effusion and mesenteric edema in these animals are likely related to cardiac changes. Slight myocarditis was also seen in one female dosed at 16 mg/kg and sacrificed as scheduled. A variety of other findings was encountered at 16 mg/kg including bile duct hyperplasia and hepatocellular hypertrophy, depletion in the lymphoid organs, increased non-lymphocytic cellularity in medullary cords in the mesenteric lymph nodes, increased metaphyseal spongiosa at the diaphysis of the femoral bone, nephropathy with glomerular and tubular alterations, increased apoptosis, mitosis and depletion of zymogen granules of acinar cells of the pancreas, interstitial edema as well as perivascular inflammatory infiltration of mesenteric fat tissue, flattened follicular epithelia in the thyroid gland. In addition, in the female genital tract the amount of large active corpora lutea in the ovaries was markedly increased whereas ovarian follicles were atrophic. Oviducts, uterus and vagina were also atrophic. In one male dosed at 16 mg/kg the rare finding of pancreatic acinar cell carcinoma was observed. In the absence of any other proliferative finding of the exocrine pancreas in this study, a spontaneous origin is most likely. Under the conditions described above and taken all together the NOAEL (no-observed-adverse-effect-level) for Bis- (2,6-diisopropylphenyl )carbodiimid is found to be 4 mg/kg for male and female rats. The dose of 16 mg/kg caused distinct toxicity including death, decreased body weight gain as well as alterations in various organs including heart, liver, kidney, white blood cells, gastro-intestinal tract, female genital tract.