1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-02 to 2011-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with GLP and OECD/EU guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted: 3. October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents, EPA Health Effects Test Guidelines, June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Incorez 397

IUPAC Name:

Incorez 397

Constituent 2

Reference substance name:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

EC Number:

619-764-7

Cas Number:

173904-11-5

Molecular formula:

C16H30N2

IUPAC Name:

(2-methylpropylidene)[(3-{[(2-methylpropylidene)amino]methyl}cyclohexyl)methyl]amine

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

other: Hsd.Brl.Han: of Wistar origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male animals: 6 – 7 weeks old
Female animals: 8 – 9 weeks old
- Weight at study initiation:
Male animals: 180 – 198 g
Female animals: 136 – 61 g
- Fasting period before study:
No data
- Housing:
2 or 3 animals of the sex/ cage
- Diet (e.g. ad libitum):
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water (e.g. ad libitum):
ad libitum
- Acclimation period:
7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (Helianthii annui oleum raffinatum)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle, in sunflower oil, in concentrations of 25 mg/mL, 125 mg/mL and 500/375 mg/mL. For the treatment of high dose animals, 500 mg/mL was used from day 0 up to day 15 and 375 mg/mL was used from day 16 onwards.

VEHICLE
- Justification for use and choice of vehicle:
The test item is not soluble in water; therefore sunflower oil (Oleum helianthi) was used for preparing formulations appropriate for oral administration. Sunflower oil was a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle:
25, 125 and 500/375 mg/L (nominal)
- Amount of vehicle: 2mL
- Lot/batch no.: 2011.03.04.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of Incorez 397 was carried out with a GC method using MS detection. For analysis of the achieved concentrations of Incorez 397 in a formulation the sample was diluted with n-Hexane. The test item was formulated in the vehicle, in sunflower oil, in concentrations of 25 mg/mL, 125 mg/mL and 500/375 mg/mL. For the treatment of high dose animals, 500 mg/mL was used from day 0 up to day 15 and 375 mg/mL was used from day 16 onwards. Formulations were prepared in the formulation laboratory of Test Facility daily. Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Samples were taken on day 4 and day 25 (June 06 and June 27, 2011repectively). The measured concentrations ranged from 87.6 to 95.8 % of nominal concentrations at the first occasion and from 71.4 to 83.0 % of nominal concentrations at the second analytical occasion. The low concentration values obtained at the end of the study was probably due to a slow decomposition of Incorez 397. The test item was stored in its original container and handled according to prescription of MSDS, however using it for a long time in studies (opening of container daily) resulted in a slow hydrolysis with humidity of air. Results of formulation analysis are attached in Appendix X.
The mean values of the concentrations measured at the two analytical occasions were calculated for the correcting real doses administered to animals.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50, 250 and 1000/750 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
39, 193 and 937/609 mg/kg bw/day
Basis:
other: analytical verification

No. of animals per sex per dose:

5 male and 5 female animals per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting was based on findings obtained in “14-Day Oral Gavage Dose Range Finding Study with Incorez 397 in the Rat” (Study no. 644.103.2618). The doses were 50, 250 and 1000 mg/kg bw/day. In this Dose Range Finding Study, 1000 mg/kg bw/day dose of Incorez 397 caused clinical observations, a slight reduction in the food consumption, body weight gain and slight changes of clinical pathology.
The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically. In case of severe signs of toxicity high dose will be reduced.
1000 mg/kg bw dose of Incorez 397 caused mortality of one male and one female animal on day 9 and 7, respectively. Therefore, the high dose was reduced to 750 mg/kg bw/day from day 16 onward. The mid and low doses remained at 250 and 50 mg/kg bw/day.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:
All animals were weighed with an accuracy of 1 g on days 0, 7, 14, 21 and 27. Animals of high dose group were measured daily from day 7 to day 16 since the body weight of animals decreased during the first week, and the treatment volume was adjusted daily to increase the accuracy of dosing. Individual body weight changes were calculated between days 0-7, 7-14, 14-21 and 21-27.
Fasted body weight was measured on day of necropsy (day 28).

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anaesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 ml 9NC Microtube, 0.106 mol/L, Sarstedt,), one for haematology (MiniCollect® EDTA tubes, spray-dried, 0.5 ml, Greiner Bio-One International AG), and the third one (VACUETTE® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.
Tubes for haematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 ml blood was collected, if possible into clinical chemistry tubes.
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anaesthesia. Three samples were taken from each animal: one for determination of blood clotting times (for APTT and PT; 1.0 ml 9NC Microtube, 0.106 mol/L, Sarstedt,), one for haematology (MiniCollect® EDTA tubes, spray-dried, 0.5 ml, Greiner Bio-One International AG), and the third one (VACUETTE® Serum Tube, 2.5 ml, Greiner Bio-One International AG) to obtain serum samples for clinical chemistry.
Tubes for haematology and coagulation were filled up to the final volume (marked on the tubes) and at least 1.0 ml blood was collected, if possible into clinical chemistry tubes.
- Animals fasted: Yes
- How many animals: all animals

NEUROBEHAVIOURAL EXAMINATION: Yes
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on all animals in the fourth exposure week. General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioural and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal irrespective of the date of mortality, either during the study or following termination of the experimental phase. Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of narcosis by Isoflurane CP® (details are presented in "Details of Other Materials").
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, colour, shape and size. The following organs/tissues were preserved in 4 % buffered formaldehyde solution except testes and epididymides, which were fixed in modified Davidson solution.
adrenals
aorta
bone marrow (femur)
brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
eyes (lachrymal gland with Harderian glands)
female mammary gland
gonads (testes with epididymides, ovaries, uterus with vagina)
gross lesions
heart
kidneys
large intestines (caecum, colon, rectum, including Peyer’s patches),
liver
lungs (with main stem bronchi; inflation with fixative and then immersion;)
lymph nodes (submandibular and mesenteric)
muscle (quadriceps)
oesophagus
pancreas
pituitary
prostate
salivary glands (submandibular)
sciatic nerve
seminal vesicle with coagulating gland
skin
small intestines (representative regions: duodenum, ileum, jejunum)
spinal cord (at three levels: cervical, mid-thoracic and lumbar)
spleen
sternum
stomach
thymus
thyroid + parathyroid
trachea
urinary bladder

The following organ weights were determined and recorded:
With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, prostate and seminal vesicles with coagulating glands;
With precision of 0.001g: adrenals, ovaries

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (group 1) and high dose (group 4) groups.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- haematology
- clinical chemistry
- organ weight data

The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.

Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The frequency of clinical symptoms and pathologic findings were calculated. Results were evaluated in comparison with values of control group (i.e. control value).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
The test item caused mortality in one male (no. 152) and one female (no. 175) animal administered with 1000 mg/kg bw/day on days 10 and 7, respectively.
After the high dose was reduced to 750 mg/kg bw/day no further mortality occurred. No mortality was observed in the control, 50 and 250 mg/kg bw/day groups during the course of study.
Daily clinical observations
Animals treated with 1000 mg/kg bw/day showed decreased activity, salivation, Straub-tail, and nuzzling up of bedding (5/5 male and 5/5 female) during daily clinical observations. Additionally, vocalization was noted for one male (1/5) and one female (1/5) rat. The first observations are noted on day 1.
After the dose reduction to 750 mg/kg bw/day (day 16), only salivation and nuzzling up of bedding were observed from day 20 up to the last treatment day. Straub tail and activity decrease ceased on day 18 and hunched back was noted for two female animals (2/4) for a day (day 19).
250 mg/kg bw/day dose of Incorez 397 caused salivation in all male (5/5) and female (5/5) animals from day 7 up to the last day of treatment with individual variations.
There were no clinical signs in the control and 50 mg/kg bw/day groups.

Detailed weekly clinical observations
No clinical signs were noted for any groups in the course of the detailed weekly observations. The general physical condition and behaviour of animals were considered to be normal throughout the entire observation period.

Functional observation battery
Positional struggle in a marked degree was found in the control
(1/5, male), in 1000 mg/kg bw/day (1/4, male) and in 50 mg/kg bw/day (1/5, female) group. The equilibrium test was negative in one male
(1/5, 50 mg/kg bw/day) and in one female (1/5, control) animal.
Functional battery observation determinations demonstrated no treatment-related differences with respect to the controls in the behaviour or in reactions to different type of stimuli at the end of the treatment period. Incidences and extent of reactions observed in groups of treated animals were comparable to those observed in control animals, and are, thus, without any toxicological significance. Variations in positional struggle and equilibrium test were within the normal biological variation, with respect to behaviour, reactions to different type of stimuli or manipulations.

In summary, Incorez 397 caused death of animals and clinical signs (decreased activity, salivation, Straub-tail, nuzzling up of bedding, and vocalization) in male and female animals after treated with 1000 mg/kg bw/day. Animals recovered up to the time of next treatment as no signs were noted at the weekly observation and during the functional observation.
750 mg/kg bw/day induced salivation and nuzzling up the bedding while other signs (decreased activity, Straub-tail, and vocalization) disappeared after the dose reduction.
At 250 mg/kg bw/day, salivation was found in all animals.

BODY WEIGHT AND WEIGHT GAIN
In male animals treated with 1000/750 mg/kg bw/day group, body weight remained below control values during the entire study due to the significantly reduced body weight gain on weeks 1 and 2. After dose reduction to 750 mg/kg bw/day, the body weight gain was similar to that in the control group, but the total body weight gain (between days 0 and 27) remained below the control value (p < 0.01, each).
For female animals treated with 1000 mg/kg bw/day, mean body weight and mean body weight gain was comparable to the control group throughout the treatment period.
The mean body weight and body weight gain were similar to those of the control group in male and female animals at 50 and 250 mg/kg bw/day throughout the treatment period.
In summary, 1000 mg/kg bw/day of Incorez 397 caused a reduction in body weight in male animals, while body weight of female animals was not influenced. During the administration of 750 mg/kg bw/day dose, there was no difference in the body weight gain of male animals with respect to control group, however total body weight gain remained significantly below the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE
In male animals treated with 1000 mg/kg bw/day, the mean daily food consumption was significantly reduced in compared to the controls during the first and second week (p < 0.01 and p < 0.05, respectively). In female animals, the daily mean food consumption was less than in the control group, however, without statistical significance.
The mean daily food consumption was similar in the control and dosed groups after reducing the high dose to 750 mg/kg bw/day.
In animals treated with 250 mg/kg bw/day and 50 mg/kg bw/day, the mean daily food intake was similar to that of the control group both for male and female animals.

In summary, treatment significantly reduced food consumption of male animals at 1000 mg/kg bw/day during the first two weeks. Food consumption of female animals in the high dose group was slightly but not significantly reduced. Finding on food consumption were in accordance with clinical observations and the body weight gain. No significant differences were found between the control and test item treated groups after the dose reduction.

HAEMATOLOGY
There were no statistically significant differences in the examined haematological parameters between the control and test item treated male animals at 1000/750, 250 and 50 mg/kg bw/day.
In female animals, statistical significances were noted for the slightly higher mean values of white blood cell count (WBC) and percent of lymphocytes (LYMPH) at 1000/750 mg/kg bw/day, and for lower percent of neutrocytes (NE) at 1000/750 and 250 mg/kg bw/day.
There were no statistically significant differences in the examined haematological parameters between the control and test item treated groups at 50 mg/kg bw/day.
In summary, haematological evaluation did not reveal test item related changes in the examined parameters. Statistically significant differences noted for white blood cell count and percent of lymphocytes at 1000/750 mg/kg bw/day and for percent of neutrocytes at 1000/750 and 250 mg/kg bw/day, were not considered to be toxicologically significant and all values were within the historical control ranges. There was no dose-response relationship.

CLINICAL CHEMISTRY
At 1000/750 mg/kg bw/day, statistical significant differences were noted for male animals in activity of alkaline phosphatase (ALP).
In female animals, concentration of glucose (GLUC), sodium (Na+), chloride (Cl-), albumin (ALB) and total protein (TPROT) was less with respect to controls.
At 250 mg/kg bw/day, all examined biochemical parameters were similar to the appropriate value in the control group (male and female animals).
At 50 mg/kg bw/day, statistical significances appeared in the slightly lower activity of alkaline phosphatase (ALP) and total bilirubin concentration in male animals. In female animals, all examined parameters were similar to the control value.
In summary, there were no test item-related differences in the serum chemistry parameters. Sporadic statistical differences (ALP, Na+, Cl-, ALB, TPROT) were considered to be of little or no biological significance. All values were without dose-response relationship and mean values were within range of historical controls. The lower mean glucose concentration of female animals treated with 1000/750 mg/kg bw/day was considered not toxicologically significant, since there were no histopathological changes to substantiate their relevance.

ORGAN WEIGHTS
At 1000/750 mg/kg bw/day, the absolute organ weights and organ weights relative to the body weight were not relevant for evaluation because of the lower mean body weight of male animals. The adrenal weight relative to brain weight was slightly above the control value.
In female animals, kidney weights (absolute and relative to the body and brain weights) were higher with respect to controls.
At 250 and 50 mg/kg bw/day, no differences in the examined organ weights were noted with respect to controls in male and female animals.
In summary, evaluation of absolute and relative organ weights did not reveal clear evidence of test item-related alterations in organ weights. Changes in adrenal (male) and kidneys (female) weight at 1000/750 mg/kg bw/day were with small degree and were not associated with any treatment-related clinical pathological or macroscopic findings and there were no supporting histopathological alterations.

GROSS PATHOLOGY
In dead animals, cannibalism autolysis of visceral organs were observed.
At 1000/750 mg/kg bw/day, pinhead sized haemorrhages (1/5, male) and pale kidneys (1/5, male), were observed at the necropsy.
At 250 mg/kg bw/day, pale kidneys were noted for one individual male animal (1/5).
In females, pale liver (1/5) and hydrometra in a slight and moderate degree (2/5) were observed.
At 50 mg/kg bw/day, reddish mottled lungs (1/5, male) and moderate hydrometra (1/5, female) were detected.
In the control group, reddish mottled lungs (1/5 male, 1/5 female), point-like haemorrhages in the lungs (1/5 male), alopecia and scar on left shoulder (1/5 female) and moderate hydrometra (2/5 female) were detected.
In summary, gross necropsy of male and female animals did not reveal any macroscopic changes which were related to treatment with test item. Pale kidneys and pale liver observed in animals treated with 250 and 1000/750 mg/kg bw/day were considered to be individual alterations. These are common findings which may also appear in untreated animals. Pulmonary changes (haemorrhages, reddish mottled colour) were due to the exsanguination procedure occurring also in untreated rats; these were without any toxicological relevance in this study. Hydrometra, related to the female sexual cycle, and alopecia and wound on the skin are frequent observations in experimental rats, which have no toxicological meaning.

HISTOPATHOLOGY: NON-NEOPLASTIC
In male animals treated with 1000/750 mg/kg bw/day, in the lungs minimal alveolar emphysema (1/4 male; 1/4 female) and minimal or mild hemorrhage (2/4 male), in the fore-stomach focal restorative inflammation (1/4 male, 2/4 female) in mild or moderate degree occurred.
At 250 mg/kg bw/day, kidneys (1/5 male) and liver (1/5 female) were processed and examined histologically following macroscopic observations. However, no lesions were found in any organs at the microscopic examination.
In the control group, lungs showed minimal alveolar emphysema (1/5 male; 2/5 female) and minimal or mild hemorrhage (1/5 male, 2/5 female). In the forestomach focal restorative inflammation at mild degree (1/5 male, 1/5 female) was observed. Additionally, changes in skin including mild focal fibroma and mild hyperkeratosis with mild lympho-histiocytic infiltration (1/5), and dilatation in uterus (1/5female) were noted in female animals.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, kidney, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system was detected. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.
The histological picture of the kidneys – including the renal corpuscles, the proximal, and distal convoluted tubules, the proximal and distal straight tubules, the collecting ducts and the pelvis - were the same, in the control and test item treated animals. No degenerative lesions, cellular infiltrates, inflammatory, hyperplastic or neoplastic lesions were visible.
In conclusion, INCOREZ 397 administered by oral gavage daily for 28 days to male and female rats at dose levels up to and including 1000/750 mg/kg bw/day did not cause any toxic or other test item related lesions detectable by histological examination of investigated organs in the experimental animal.
The higher kidney weights in female animals administered with 1000/750 mg/kg bw/day were considered as adaptation phenomenon in connection with the long term effect of test item without pathological lesion.
Pulmonary changes (focal alveolar emphysema and focal hemorrhage) occurred with similar incidence in the control and test item treated group and were related to hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The focal restorative inflammation in the wall of fore-stomach in mild or moderate degree was in connection with a mechanical injury due to the using of oral gavage.
The dilatation of the uterine horns in some control female animals was a slight neuro-hormonal phenomenon in connection with the sexual function – estrus phase – of the inner genital organs.
Histological lesions in skin (focal fibrosis, hyperkeratosis and focal lympho-histiocytic infiltration in the dermis) were considered to be the consequence of an individual mechanical skin lesion in one control female animal.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

609 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the observations and measured dose concentrations the NOAEL and NOEL for Incorez 397 in a 28-day oral toxicity study were determined as follows:
NOAEL: 609* (750) mg/kg bw/day (male and female animals)
NOEL: 39* (50) mg/kg bw/day (male and female animals)
* value corrected due to analytical results

Executive summary:

The aim of this 28-Day toxicity study was to obtain first information on the toxic potential of Incorez 397 in rats at three dose levels following 28-day oral administration.

The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=5 animals/sex/dose) once a day at 0 (vehicle control), 50, 250 and 1000/750 mg/kg bw/day nominal doses (low, mid and high dose) corresponding to nominal concentrations of 25, 125 and 500/375 mg/mL, applied in a dose volume of 2 mL/kg bw for 28 days. Stability, concentration and homogenous distribution of test item in sunflower oil were confirmed analytically beforehand. Incorez 397 proved to be stable in sunflower oil at room temperature for 24 hours. Incorez 397 content of formulations administered to animals ranged from 87.6 to 95.8 % of nominal concentrations at the first occasion and from 71.4 to 83.0 % at the second analytical occasion. Calculating with the measured concentrations, the real doses administered were as follows: 39, 193 and 937/609 mg/kg bw/day respectively to nominal doses of 50, 250 and 1000/750 mg/kg bw/day. The results of study are reported referring to the initial nominal concentrations. The NOAEL was determined regarding the analytical differences of nominal concentrations.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted on the last week of the study. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations including haematology, and clinical chemistry were conducted at the termination of the treatment. Gross pathology was conducted on all animals on the day following the last treatment. The absolute and relative organ weights of selected organs were determined. A full histopathology examination was performed on the preserved organs and tissues of the animals of control and high dose groups and on organs with macroscopic findings in the low dose groups.

The results were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle (sunflower oil) only.

 

Results:

 

Mortality

One male (1/5) and one female (1/5) animal of the high dose group were found dead on days 10 and 7, respectively.

 

Clinical observations

In the high dose group, decreased activity, salivation, Straub-tail, and nuzzling up of bedding were observed in each male and female animal. Additionally, hunched back and vocalization were noted for some rats. After dose reduction, only salivation and nuzzling up of bedding were observed from day 20 up to the last treatment day. 250 mg/kg bw/day caused salivation in male and female animals.

No toxic signs related to the test item were found at any dose level at the detailed weekly clinical observations and the functional observation battery, indicating animals recovered quickly from signs caused by the daily treatment.

 

Body weight and body weight gain

The body weight gain was reduced at 1000 mg/kg bw/day (male and female animals) during the first two weeks, resulting in lower body weight only in male animals during the entire observation.

 

Food consumption

The food consumption was decreased during the first week (male and female) and during the second week (male) at 1000 mg/kg bw/day.

 

Clinical pathology

There were no test item related alterations in the examined haematology and clinical chemistry parameters.

 

Organ pathology

Specific macroscopic alterations related to treatment with the test item were not found during the terminal necropsy.

 

No test item related organ weight changes were found after the 14-day treatment period.

Histopathological investigations did not reveal specific test item related alterations at 1000/750 mg/kg bw/day.

Under the condition of the present study, Incorez 397 caused death and clinical signs (mainly decreased activity, Straub tail, salivation and nuzzling up of bedding material) in male and female animals at a dose of 1000 mg/kg bw/day in Hsd.Brl.Han: Wistar rats. In accordance with the clinical observations, a test item influence on the food consumption and body weight was found at 1000 mg/kg bw/day.

750 mg/kg bw/day induced only clinical signs (salivation and nuzzling up of bedding).

At 250 mg/kg bw/day salivation appeared in male and female animals.

Based on these observations and measured dose concentrations the NOAEL and NOEL for Incorez 397 in a 28-day oral toxicity study were determined as follows:

NOAEL: 609* (750) mg/kg bw/day (male and female animals)

NOEL: 39* (50) mg/kg bw/day (male and female animals)

* value corrected due to analytical results