Registration Dossier

Administrative data

Description of key information

1. Subacute oral repeated dose (2012), 28 days, rats, gavage, NOAEL 4mg/kg bw;
2. Subacute oral repeated dose (1962), 28 days, rats, gavage, NOAEL 8 mg/kg;
3. Reproduction/ Developmental Toxicity Screening Test; rats, gavage, NOAEL 3 mg/kg bw.


The substance meets the criteria for classification and labelling into the STOT-RE Cat 1 (H372).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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
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
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
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.

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.
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality (two reliable GLP guideline studies: 28-day study and Screening Test).
Organ:
blood
heart
kidney
liver
ovary
uterus
vagina

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Subacute oral repeated dose study in rats (2012)

Bis- (2,6-diisopropylphenyl )carbodiimide 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 haematology 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 oedema 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 oedema 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)carbodiimide 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 and female genital tract.

Subacute oral repeated dose study in rats (1962)

A subacute oral repeated dose study was undertaken in rats with the test item bis(2,6-diisopropylphenyl)carbodiimide (1962). This experiment was done similar to the OECD guideline 407 (1962). The test material was administered by gavage to four groups of male rats for twenty-eight days (5 days per week), at the dose levels of 0, 8, 16 and 32 mg/kg/day. A control group was dosed with vehicle alone (sesame oil). After the dosing period the animals were maintained without treatment for a further 28 days. Clinical signs, bodyweight development and functional observations were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues. Mortality occurred in the highest dose group (32 mg/kg bw, 90 %) and in the middle dose group (16 mg/kg bw, 30 %).The general condition of the animals in the 32 mg/kg bw dose group was greatly impaired after only a few doses. The animals became very weak, lost weight, and became so susceptible to infections, before they died of contracted pneumonia. The microscopic examination of the remaining deceased rat revealed toxic damage to the myocardium; this could, however, have been caused by bacteria toxins. Damage to the liver and kidneys was seen additionally in some of the animals. The liver damage was also seen in the one rat which survived the twenty doses and was then killed. In the 16 mg/kg bw dose group, the general condition of the animals was impaired within a very short time. Pneumonia was the cause of death, additionally damage to the liver and kidneys was found. The urinalyses and blood counts of the surviving animals were normal. Damage to the liver and kidneys was also found in some of the animals killed after the end of the experiment. Organ weight data show that the liver and kidney weights of these animals were elevated. Increased hepatic activity of this type is often observed in the rodent liver following the administration of xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction. No mortality or toxicologically significant clinical observations were detected in the animals treated with 8 mg/kg bw. This daily dose was tolerated by the rats without symptoms, body weight gain was unchanged and they also behaved normally throughout the subsequent 4-week observation period. The organs of the animals killed at the end of the experiment four weeks later were normal. The blood counts and urinalyses gave no indication of any damage to the kidneys or bone marrow. A “No Observed Effect Level” (NOEL) has been achieved. 8 mg/kg/day were given as a “No Observed Adverse Effect Level” (NOAEL).

Reproduction/ Developmental Toxicity Screening Test in Rats (2013)

Bis-(2,6-diisopropylphenyl) carbodiimide was administered daily via gavage in corn oil as vehicle to 12 male and 12 female Wistar rats per dose group, in doses of 0, 1, 3 or 8 mg/kg body weight (Popp, 2013; Study No. T6084004). Dosing of the highest dose group treated with 8 mg/kg bw was stopped on study day 8, as toxicity in this dose was too pronounced for the purpose of the study. Animals of both sexes were not treated on study day 9 and 10. In the morning of study day 11 dosing was continued at a reduced dose of 5 mg/kg bw.

Treatment started 2 weeks prior to mating and continued during the mating period of up to 2 weeks. Males were dosed further up to necropsy for a total period of at least 4 weeks and females were dosed during gestation and lactation up to their necropsy on day 4-6 p.p.

Investigations were performed on general tolerance of the test compound by the parental animals as well as on effects on reproduction including early postnatal development of F1 pups. The animals were regularly observed and weighed, food intake and reproduction parameters were determined. Selected organs were weighed and organs were subjected to macroscopical and histopathological investigations.

The test substance was stable in the vehicle for the duration of use. Formulations given to the rats were prepared appropriately.

Survival of both sexes was not affected in the groups treated with 1 or 3 mg/kg bw. At the high dose of 8 mg/kg bw one female (No. 91) died on study day 8 and one female (No. 90) was killed moribund on study day 11 after a two days drug holiday. The high dose was reduced to 5 mg/kg bw after the two days of drug holiday. With that dose there was no obvious clinical toxicity and no further mortality.

Clinical observations did not reveal any relevant adverse effects in males up to the high dose and in females up to the mid dose of 3 mg/kg bw An impaired general condition was observed in females starting at 8 (5) mg/kg bw. During premating and mating in-cage observations the females of this dose group revealed piloerection, a squatting position and accelerated breathing after dosing. Furthermore, sunken flanks, a hunched back, high-stepping gait, an increased respiratory rate, piloerection and paleness were noted. The females of the high dose group without litter presented piloerection at daily clinical observation and cage-side observation after treatment. None of the high-dosed females was pregnant or littered.

Body weight was reduced in males of the highest dose group. High-dosed females which revealed a decrease in bw on day 8 of premating, recovered after the two days of drug holiday and a reduction to 5 mg/kg bw. The reduction of food intake observed in high-dosed females and males in the first week of premating was comparable in all groups during the second week of the premating period. Food intake was also reduced in females of the 3 mg/kg bw dose group during first week of gestation, but no effect on food consumption was seen during the second and third week of gestation and during the lactation period, anymore. (No pregnancy could be observed in the high dose group).

Necropsy revealed an enlarged adrenal gland, a slightly collapsed, dark-redded lung and aqueous liquid in the chest cavity of the high-dose female No. 91, which died on study day 8.

High-dosed female No. 90, which was killed moribund after a two days drug holiday on study day 11, presented a changed surface of kidneys, a smaller thymus and a red, elastic lump at the mucosa of the gastro-esophageal vestibule at necropsy.

Histopathology of No. 91 revealed a slight myocardial degeneration, an alveolar edema in the lungs, slight multifocal bile duct hyperplasia, and signs of moderate tubular degeneration and regeneration in the kidneys and No. 90 presented a marked ulcer in the forestomach.

During terminal necropsy, no implantation sites and no corpora lutea were recorded in the ovaries in any of the animals from the high dose group. This has to be attributed clearly to the treatment with test article. In contrast, the distribution of the number of implantation sites and corpora lutea was comparable among the controls and the animals treated with 1 or 3 mg/kg bw/day body weight test substance. No further histopathological, treatment-related and toxicologically relevant findings could be observed up to the highest dose group.

The insemination index was slightly reduced in high-dosed rats. Additionally fertility and gestation were severely affected in the high dose. No gestation could be observed in this group, which has to be attributed clearly to the treatment with the test article. No implantation sites and no pups were seen in high-dosed females. At 3 mg/kg bw a slight, but statistically not significant increase of prenatal losses was observed.

No toxicologically relevant clinical signs were observed in F1 pups during lactation. One pup of dam No. 82 of the 3 mg/kg bw dose group died on day 0 and revealed no milk in it´s stomach; the other pup of this dam died on day 1 and was found in an autolytic state. 

Overall, a steep dose response curve was observed and the following no-observed-(adverse)-effect-levels were determined: NOAEL of 3 mg/kg bw for F0 rats (general toxicity) and NOEL of 1 mg/kg bw for reproduction and developmental toxicity, due to the slight increase of prenatal losses at 3 mg/kg bw.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The available subacute repeated dose toxicity study is reliable and sufficient for an assessment of repreated dose toxicity of the target substance. As the results of the 28-day repeated dose study (Popp, 2012) show toxic effects, which lead to the classification as STOT-RE Cat. 1, an additional testing for subchronic repeated dose toxicity is not indicated for this substance and therefore waived.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: heart; cardiovascular / hematological: hematopoiesis; cardiovascular / hematological: lymph nodes; digestive: pancreas; urogenital: kidneys; urogenital: ovaries; urogenital: uterus; urogenital: vagina

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008, the test material meets the criteria for classification and will require labelling for systemic organ toxicity after repeated exposures (STOT-RE) due to the following reasons:

The established NOAEL of 4 mg/kg bw in the 28 -day study and the effective dose level of 16 mg/kg bw (the effective dose level is the lowest dose inducing significant/severe target organ toxicity) are both below the GV (Guidance value), which triggers C&L. Similarly, NOAEL of 3 mg/kg bw was established in the OECD 421 study; the effective dose was 8(5) mg/kg bw. The GV for Cat I is ≤ 30 mg/kg bw and for Cat II is ≤ 300 for 28-day study and for oral route of exposure (Table 3.9.2.2. in Section 3.9.2 “Classification of substances for STOT-RE” in Guidance on the Application of CLP criteria “Guidance on Application of Regulation (EC) No.1272/2008”);

In the 28 -day study, the test substance induced mortality and caused poor general condition in the treated animals. The mortalities clearly resulted from repeated exposure since two animals died on day 11 and one animal on day 29. Clinical signs observed in the high dose group of animals were not limited to the first days of treatment but started in the week 2 of treatment. According to the study director, the mortalities are likely related to cardiac changes: myocardial degeneration/myocarditis associated with atrial thrombosis. Gross pleural effusion and mesenteric oedema in these animals is likely related to cardiac changes. Body weight was significantly decreased in the animals of the high-dose group and a severe decrease was observed especially in females (-28.7 and -81.7% for body weight and body weigh gain, respectively). There were adverse changes in haematology and significant organ damage was noted at necropsy, which was confirmed by histopathology. The following target organs were identified in the study:

Liver: Hyperplasia of bile duct and hepatocellular hypertrophy;

Lymphoid organs: Depletion in various organs and increased non-lymphocytic cellularity in medullary cords of the mesenteric lymph nodes;

Femoral bone: Increased metaphyseal spongiosa at the diaphysis;

Kidney: Nephropathy with glomerular and tubular alterations;

Pancreas: Increased apoptosis, mitosis and depletion of zymogen granules of acinar cells. This finding might be the reason for the distinctly lower body weight gain at the high dose;

Mesenteric fat tissue: Interstitial oedema as well as perivascular inflammatory infiltration;

Thyroid gland: Flattened follicular epithelia (not associated with any changes in thyroid hormones);

Female genital tract: marked increase in the amount of large active corpora lutea in the ovaries, atrophy of ovarian follicles, atrophy of oviducts, uterus and vagina.

According the conclusion of the study report, target organs are mainly the heart, white blood cells and lymphoid organs, gastro-intestinal tract, liver, kidney, and female genital tract.

 

The results of the 28 -day study are confirmed by the outcome of the OECD 421 study. There were deaths and poor general condition of females dosed with 8 mg/kg bw (the highest dose). The dose was therefore reduced to 5 mg/kg bw on day 11 of treatment. None of the high-dosed females was pregnant or littered. Furthermore, body weight and food intake were reduced in the first week of premating (at 8 mg/kg bw). The target organs were similar to those in the 28 -day study. Gross necropsy of females that died revealed changed surface of kidneys, smaller thymus, a red, elastic lump at the mucosa of the gastro-esophageal vestibule, an enlarged adrenal gland, a slightly collapsed, dark-reddened lung and aqueous liquid in the chest cavity. Histopathology revealed a slight myocardial degeneration, an alveolar edema in the lungs, slight multifocal bile duct hyperplasia, and signs of moderate tubular degeneration and regeneration in the kidneys as well as a marked ulcer in the forestomach. During terminal necropsy, no implantation sites and no corpora lutea were recorded in the ovaries in any of the animals from the high dose group of 8(5) mg/kg bw.

 

In both studies, hyperplasia of bile duct was observed in animals that died. Hepatocellular hypertrophy was observed in animals of the high dose group in the 28 -day study. Generally, hepatocellular hypertrophy is an adaptive response to exposure to a chemical. This is well known for rodents and not relevant for humans. Slight increase in alanine aminotransferase (ALAT) was found in males and a slight increase in higher alkaline phosphatase (APh) was found in both sexes. These findings point to a certain liver toxicity. The liver weights (absolute and relative) were, however, not affected. The liver weight was, however, statistically significant increased in the high-dosed female rats in the Screening Test. Additionally, no liver cells necrosis is reported in both studies. Based on these results, liver is considered possibly not to be a target organ. Regarding thyroid toxicity in rodents observed in the 28 -day study, it is normally associated with a reduction in thyroid hormone levels. However, the changes seen in thyroid glands were not associated with any changes in thyroid hormones and can be caused by other mechanisms. Nephropathy is a well known effect for male rats, however, nephropathy was observed also in female animals (in both 28 -day and in OECD 421 study) and therefore kidney is considered to be a target organ for this chemical. Regarding female genital tract, similar effects were found in both studies. The substance produced marked increase in the amount of large active corpora lutea in the ovaries, atrophy of ovarian follicles, and atrophy of oviducts, uterus and vagina (28-day study). At the high dose of 16 mg/kg (28-day study), absolute and relative weights of ovaries and uterus were reduced in females. In the OECD 421 study, total loss of implantation (no implantation sites, no corpora lutea) was observed in the high dose group (8(5) mg/kg bw). Based on these results, female genital tract is considered to be a target organ for this substance. The latter however will not contribute to a classification as STOT RE, but rather to reproductive toxicity.

 

Conclusion

According to Annex I, Section 3.9.1.1. of the CLP Regulation, classification of the substance for specific target organ toxicity – repeated exposure is indicated when there is specific target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. The above effects demonstrate adverse effects on various organs.

Under Section 3.9.2.9.5 of Annex I of the CLP Regulation, guidance value of 10 mg/kg body weight per day in a 90-day oral rat study corresponds to 30 mg/kg body weight per day in a 28-day oral rat study. The effects were demonstrated at or below 16 mg/kg bw/d.

As a classification into category 1(regarding systemic organ toxicity) should not be based only on the guidance values given, but additional human data, several animal data and the severity of the effects should be taken into account. Only very severe effects do trigger category 1 classification, other significant effects trigger the category 2. Taking into account the grade of the severity of adverse effects in the treated animals (the effects observed in the 28-day and in the OECD 421 studies are significant but mostly not severe (males were much less affected than females)), the chemical may also only require classification for systemic organ toxicity after prolonged exposures (STOT-RE) - Cat 2 (H373 - May cause damage to organs through prolonged or repeated exposure).

The overall constellation of observed effects and their severity and so the necessity of classification STOT RE, Cat. 1 may be hence considered as a borderline case. Nevertheless, out of precautionary reasons, classification as STOT Rep. Exp. 1 (H372 - Causes damage to organs through prolonged or repeated exposure), will be done. Excluding species-specific effects in target organs and organs covered by other classifications, the following organs are considered to be target organs for classification and labelling: the heart, white blood cells and lymphoid organs, gastro-intestinal tract, and kidney.