9-Octadecenoic acid (Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

enhanced combined repeated dose toxicity study with reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

exceeding the requirements of OECD TG 422 (e.g. test group size)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol

EC Number:

272-379-2

EC Name:

9-Octadecenoic acid (Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol

Cas Number:

68815-51-0

Molecular formula:

C18 H34 O2 . C4 H12 N2 O

IUPAC Name:

2-(2-aminoethylamino)ethanol; (Z)-octadec-9-enoic acid

Details on test material:

- Name of test material (as cited in study report): Kerocom F 100
- CAS No.: 68815-51-0
- Physical state: solid (melt)/brown
- Analytical purity: 100% CAS 68815-51-0 (9-Octadecenoic acid (9Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol)
- Lot/batch No.: 09001529U0
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed (Expiry date: 09 May 2010)
- Storage condition of test material: Room temperature, temperatures > 50 °C were avoided

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 11-13 weeks
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat
- Water (e.g. ad libitum): Drinking water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was liquefied at a temperature of about 40°C in a drying chamber. Thereafter, the specific amount of test substance was weighed, topped up with olive oil in a beaker and intensely mixed with a magnetic stirrer. During administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guarantee that the test substance concentrations in the vehicle will remain stable. The test substance preparations were stored at room temperature.


VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days in males; 48 days in females

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- After a 14-day premating period, the male and female parental animals were mated overnight in a 1:1 ratio until there is evidence of copulation or for a maximum period of 14 days has been elapsed.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical observations (DCO) were performed in 10 parental males and females per group once before the first administration and thereafter at weekly intervals.
- Observations: Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/consistency), Assessment of the urine discharged during the examination, Pupil size


BODY WEIGHT: Yes
- Time schedule for examinations: once a week


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: once a week


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Anaesthetic used for blood collection: Yes (Isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: 12 parental males and females (with litter) per group
- Parameters checked: Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Differential blood count, Reticulocytes, Preparation of blood smears (only evaluated blood smears will be archived), Prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Yes
- How many animals: 12 parental males and females (with litter) per group
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Serum glutamyl transferase, Sodium, Potassium, Chloride, Inorg. phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Magnesium


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume, Color, Turbidity, pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Microscopy of sediment


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Functional observation battery (FOB) was carried out in 5 parental males and females (with litter). The FOB was carried out in a randomized sequence. The animals were not transferred to new cages, and feed or drinking water was not be withdrawn before the tests.
1. Home cage observation: The animals were observed in the rack for a short period (about 10-30 seconds) in their cages with the lids closed, while disturbing influences (touching of the cage and loud noises) were avoided. Besides other abnormalities, particularly the following parameters were observed: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities
2. Open field observation: The animals were removed from their cages by the investigator and placed in a standard arena (50 x 50 x 25 cm). Besides other abnormalities, the following parameters were assessed: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypies, Gait abnormalities, Activity/arousal level, Feces excreted within 2 minutes (number of scybala discharged/appearance/ consistency), Urine excreted within 2 minutes (amount/color), Rearing within 2 minutes.
3. Sensory motor tests/Reflex tests: The animals were removed from the open field and were subjected to the sensory motor and reflex tests listed: Behavior when an object is approached (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, pain perception (Tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test
4. Motor activity measurement (MA): The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. The animals were placed in new clean polycarbonate cages for the time of measurement. 18 beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes in each case. The animals were given no feed or water during the measurements.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All parental animals were sacrificed under Isoflurane anesthesia by decapitation and were exsanguinated. The animals were necropsied and assessed by gross pathology, with particular attention on the reproductive organs. Animals which die intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology. The weights of following organs were determined: Anesthetized animals, Liver, Kidneys, Adrenal glands, Testes, Epididymides, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Thymus, Spleen, Brain, Heart, Thyroid glands (with parathyroid glands). The following organs or tissues of the parental animals were fixed in 4% formaldehyde solution or in modified Davidson’s solution: All gross lesions, Brain, Spinal cord (cervical, thoracic and lumbar cord), Sciatic nerve, Pituitary gland, Salivary glands (glandula mandibularis and glandula sublingualis), Thyroid glands (with parathyroid glands), Adrenal glands, Prostate gland, seminal vesicles, coagulation glands, Ovaries (fixed in modified Davidson's solution), Uterus, oviducts, vagina, Testes (fixed in modified Davidson's solution), Epididymides (fixed in modified Davidson's solution), Female mammary gland, Thymus, Lymph nodes (axillary and mesenteric), Spleen, Trachea, Lungs, heart, Aorta, Liver, Pancreas, Kidneys, Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum (with Peyer’s patches), ileum, Cecum, colon, rectum, Urinary bladder, Sternum with marrow, Larynx, Pharynx, Nose (nasal cavity), Bone marrow (femur), Eyes with optic nerve, Femur with knee joint, Skin, Skeletal muscle

Statistics:

Dunnett test (two-sided) was performed for food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter. Fisher's exact test was used for number of live and dead pups and different indices (e.g. mating index, fertility index, gestation index) and number of litters with necropsy findings in pups. Wilcoxon test (one-sided) was used for proportion of pups with necropsy findings per litter. Kruskal-Wallis test (two-sided) and Wilcoxon test was used for feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity.

Kruskal-Wallis and Wilcoxon-Test was performed for clinical pathology parameters (except differential blood count and reticulocytes), weight of the anesthetized animals and absolute and relative organ weights. Fisher's test was used for Urinalysis (except for volume, color, turbidity and specific gravity).

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups.
Clinical observations for males and females (except gestation and lactation periods): All male and 17 female F0 parental animals of the 1000 mg/kg bw/d-group, 24 male and 2 female animals of the 300 mg/kg bw/d-dose group and one F0 male animal of the 100 mg/kg bw/d-group showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes). This symptom was initially observed during study week 0. The temporary salivation is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals at the 10 mg/kg bw/d dose level. One F0 parental female of the high-dose group (1000 mg/kg bw/d) showed a skin lesion on its left flank during study week 7. This finding is considered to be incidental.
Clinical observations for females during gestation of F1 litter: At a dose level of 1000 mg/kg bw/d) 22 out of 25 F0 females and at a dose level of 300 mg/kg bw/d) 4 out of 25 F0 females (300 mg/kg bw/d) showed transient salivation during major parts of gestation period (GD 0 – 21 in test group 4, GD 4 – 9 in test group 3). Two low-dose F0 females (10 mg/kg bw/d) were not sperm-positive. However, one of these females had pups, giving evidence for successful copulation. Furthermore, one sperm-positive F0 female of test group 1 (10 mg/kg bw/d), three of test group 3 (300 mg/kg bw/d) and one of test group 4 (1000 mg/kg bw/d) did not deliver any F1 pups. There were no clinical findings in F0 females during the gestation period at the 10 and 100 mg/kg bw/d dose levels.
Clinical observations for females during lactation of F1 litter: In the high-dose group (1000 mg/kg bw/d), 12 out of 24 F0 females showed transient salivation during PND 0 – 4. There were no test substance-related clinical findings in F0 females at the 10, 100 and 300 mg/kg bw/d dose levels. One low-dose F0 female (10 mg/kg bw/d) nursed its pups not properly on PND 1 and 2. As a consequence, all pups of this litter showed hypothermia and were cannibalized on PND 3 at the latest. This single finding is not considered to be related to the test substance.
Detailed clinical observations: The detailed clinical observations, which took place in study weeks 0-4 in all parental animals and, additionally, in weeks 5-7 just in females, did not reveal any abnormalities in the animals of all test groups (0, 10, 100, 300 and 1000 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and mean body weight gain of the F0 males in test groups 1-4 (10, 100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study. Mean body weights of the F0 females were comparable to the controls throughout the entire study at all dose levels (10, 100, 300 and 1000 mg/kg bw/d). Females at the 300 and 1000 mg/kg bw/d dose levels gained more weight than the controls during gestation, although the increase was significant only at 1000 mg/kg bw/d during GD 7-14. On the other hand, these females lost previously gained surplus weight within days after parturition (statistically significant), which resulted in comparable body weights of control, 300 and 1000 mg/kg bw/d dose groups on PND 4. Thus, an association of this up and down of body weight change to treatment is not assumed. Mean body weight gain of the 100 and 1000 mg/kg bw/d females was temporarily increased during premating week 1-2, which are also considered to be spontaneous findings. Mean body weight gain of the 10 mg/kg bw/d and 100 mg/kg bw/d (apart from the incidental increase week 1-2) groups was comparable to the control group throughout the entire premating, gestation and lactation periods.

FOOD CONSUMPTION
Food consumption of F0 males was statistically significantly increased during premating week 1-2 at the 300 and 1000 mg/kg bw/d dose levels while it was comparable to the controls during the entire study at the 10 and 100 mg/kg bw/d dose levels. The slight and temporary increase in test groups 3 and 4 is not considered to be treatment-related. Food consumption of F0 females was statistically significantly increased during premating week 1-2 at the 1000 mg/kg bw/d dose level, while it was comparable to the controls during remaining premating, gestation and lactation. Food consumption of F0 females was comparable to the controls during the entire study at the 10, 100 and 300 mg/kg bw/d dose levels. The slight and temporary increase in test group 4 is not considered to be treatment-related.

HAEMATOLOGY
In male and female rats of test group 4 (1000 mg/kg bw/d) red blood cell (RBC) counts as well as hemoglobin values were slightly decreased. Additionally, in males of test group 2 (100 mg/kg bw/d) mean hemoglobin concentrations (MCHC) were higher compared to controls and relative reticulocyte counts were lower in males of test group 3 and 4 (300 and 1000 mg/kg bw/d). In females of test group 3 (300 mg/kg bw/d) hematocrit values were lower compared to controls. All these alterations were marginal, not dose-dependent and were not seen in the other sex. Therefore, they were regarded as incidental and not treatment-related. In males of test groups 3 and 4 (300 and 1000 mg/kg bw/d) the absolute eosinophil counts were slightly higher compared to controls. This increase was neither accompanied by any change of other differential blood cell counts nor by an increase of the total white blood cell (WBC) counts. Even in females no alteration in the differential blood cell counts occurred. Therefore, higher absolute eosinophil counts in males of the mentioned dose groups were regarded as incidental rather than treatment-related.

CLINICAL CHEMISTRY
In male and female rats of test group 4 (1000 mg/kg bw/d) alanine aminotransferase (ALT) activities and globulin values were increased. Additionally, in males of test group 4 (1000 mg/kg bw/d) albumin and the total protein levels as well as cholesterol concentration were increased. In males of test groups 2 and 3 (100 and 300 mg/kg bw/d) there were also measured higher albumin and total protein levels compared to controls. These were the only changed liver and kidney related parameters in males of these dose groups. Moreover, the total protein level is dependent on the albumin representing one fraction of the total protein. Therefore, although a treatment-related effect on these parameters cannot be excluded it was not regarded as an adverse effect in these dose groups (ECETOC Technical Report No. 85, 2002). In females of test group 2 (100 mg/kg bw/d) ALT activities were lower compared to controls. This decrease was marginal and not dose-dependent and it was in contrast to increased ALT measured in the high dose group rats. Therefore, the decrease of the ALT activities in females of test group 2 was regarded as incidental rather than treatment-related.

URINALYSIS
In males of test groups 3 and 4 (300 and 1000 mg/kg bw/d) were found higher incidences of blood in the urine samples. In females of test group 4 (1000 mg/kg bw/d) fewer crystals (above all fewer triple phosphate crystals) were observed in the urine sediment.

NEUROBEHAVIOUR
Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups.
Motor activity measurement: There were no significant deviations of motor activity in the male and female animals of all treated groups (10, 100, 300 and 1000 mg/kg bw/d) in comparison to the concurrent control group.

ORGAN WEIGHTS (for details see tables below)
The relative thymus weights were decreased in females of test groups 3 (300 mg/kg) and 4 (1000 mg/kg). Because there were no histopathological findings, a substance-related effect seems rather unlikely. Because there was no dose-response relationship and because there were no histopathological correlates, the increased kidney weights in females of test groups 2, 3 (absolute and relative) and 4 (relative) are considered incidental. The decreased spleen weights in males of test groups 1 (absolute and relative) and 3 (relative) as well as the increased absolute and relative weights of adrenal glands in males of test group 1 are considered incidental.

GROSS PATHOLOGY
All gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups. They are considered to be incidental.

HISTOPATHOLOGY PARENTAL ANIMALS (for details see tables below)
Mesenteric lymph nodes: Sinus histiocytosis was observed in the mesenteric lymph nodes of some treated males and females. The sinusoids were filled with a various number of macrophages containing foamy cytoplasm. The occurrence of sinus histiocytosis in the mesenteric lymph node might be caused by phagocytosis of the test substance and due to the lack of cytotoxicity is considered as non adverse.
Lungs: Two different findings were seen in the lungs, a granulomatous inflammation considered as treatment-related and spontaneous focal alveolar histiocytosis. A minimal to moderate granulomatous inflammation was observed in the lungs of some treated males. In all affected animals, the granulomatous inflammation was noted only in one of two investigated lobes (left lobe and right caudal lobe). The affected males showed few to multiple granulomatous foci in the left lobe (except of one animal: right caudal lobe), mainly near the bronchi and their branches. This distribution pattern indicates that after retraction of the gavage tube during gavage, small amounts of the test substance/ olive oil preparation might have been regurgitated, aspirated and finally caused the granulomatous inflammation. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells. The alveolar walls were lined by large cuboidal cells, partly with prominent nuclei. The cytoplasm of these alveolar epithelial cells was vacuolated or basophilic. Taken together, the occurrence of the granulomatous inflammation is considered as a direct and local effect of the test substance caused by gavage procedure. As consequence of a systemic toxic effect not only one lung lobe should be affected as seen in this study.
The granulomatous inflammation had to be differentiated from focal alveolar histiocytosis. The alveolar histiocytosis was characterized by variable degrees of intra-alveolar aggregation of macrophages containing foamy cytoplasm. In few males, the alveolar histiocytosis was associated with few inflammatory infiltrates. The incidence of alveolar histiocytosis was slightly increased in males of test group 4 (1000 mg/kg bw/d). Because there was no dose-response relationship and because the foci of alveolar histiocytosis occurred only singly (grade 1), a treatment-related effect seems rather unlikely.
All additional findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general, systemic toxicity

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Absolute organ weights:

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly increased or decreased (printed in bold):

 

	Male animals				Female animals
Test group (mg/kg body weight/day)	1 (10)	2 (100)	3 (300)	4 (1000)	1 (10)	2 (100)	3 (300)	4 (1000)
Adrenal glands	114%**	102%	105%	105%
Kidneys					100%	106%**	106%*	104%
Spleen	89%*	96%	90%	94%

* : p ≤ 0.05, **: p ≤ 0.01

 

Relative Organ Weights:

When compared to control group 0 (=100%), the following mean relative weights were significantly increased or decreased (printed in bold):

 

	Male animals				Female animals
Test group (mg/kg body weight/day)	1 (10)	2 (100)	3 (300)	4 (1000)	1 (10)	2 (100)	3 (300)	4 (1000)
Adrenal glands	114%**	102%	105%	106%
Kidneys					103%	107%**	108%**	106%**
Spleen	89%*	96%	89%*	94%
Thymus					101%	101%	90%*	89%*

* : p <= 0.05, **: p <= 0.01

 

Incidence and severity of Sinus histiocytosis observed in the mesenteric lymph nodes of some treated males and females:

 

	Male animals					Female animals
Test group (mg/kg body weight/day)	0	1 (10)	2 (100)	3 (300)	4 (1000)	0	1 (10)	2 (100)	3 (300)	4 (1000)
Number of animals	25	25	25	25	25	25	25	25	25	25
Sinus histiocytosis	0	0	0	0	13	0	0	0	12	25
Grade 1					9				5	1
Grade 2					4				4	5
Grade 3									3	11
Grade 4										8

 

Incidence and severity of granulomatous inflammation in the lungs:

 

	Male animals
Test group (mg/kg body weight/day)	0	1 (10)	2 (100)	3 (300)	4 (1000)
Number of animals	25	25	25	25	25
Granulomatous inflammation	0	0	1	3	10
Grade 1			1	1	1
Grade 2				2	4
Grade 3					5

 

Incidence of alveolar histiocytosis:

 

	Male animals
Test group  (mg/kg body weight/day)	0	1 (10)	2 (100)	3 (300)	4 (1000)
Number of animals	25	25	25	25	25
Alveolar histiocytosis	4	6	4	5	9

 

 

Applicant's summary and conclusion

Conclusions:

The NOAEL for general, systemic toxicity of the test substance is 1000 mg/kg body weight/day for the F0 parental animals, the highest dose tested. The NOEL (no observed effect level) is 10 mg/kg bw/d for the F0 parental rats based on treatment-related, local and non-adverse effects such as sinus histiocytosis in the mesenteric lymph nodes at 300 and 1000 mg/kg bw/d and granulomatous inflammation of one lung lobe with no corroborative clinical or pathological findings indicative of systemic toxicity at 100, 300 and 1000 mg/kg bw/d.

Executive summary:

The study exceeded the requirements of OECD TG 422 and was performed in compliance with GLP.

Kerocom F 100 was administered orally via gavage to groups of 25 male and 25 female Wistar rats (F0 animals) at doses of 10, 100, 300 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle (Olive oil Ph. Eur./DAB). The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

The following test substance-related adverse effects/findings were noted in F0 parental animals:

 

Test group 4 (1000 mg/kg bw/d)

- Minimal to slight sinus histiocytosis in the mesenteric lymph nodes of 13 males and minimal to severe sinus histiocytosis in the mesenteric lymph nodes of all females

- Minimal to moderate granulomatous inflammation in the lungs of 10 males

 

Test group 3 (300 mg/kg bw/d)

- Minimal to moderate sinus histiocytosis in the mesenteric lymph nodes of 12 females

- Minimal to slight granulomatous inflammation in the lungs of 3 males

 

Test group 2 (100 mg/kg bw/d)

- Minimal granulomatous inflammation in the lung of 1 male

 

Test group 1 (10 mg/kg bw/d)

- No test substance-related adverse findings

 

Conclusion: The NOAEL for general, systemic toxicity of the test substance is 1000 mg/kg body weight/day for the F0 parental animals, the highest dose tested. The NOEL (no observed effect level) is 10 mg/kg bw/d for the F0 parental rats based on treatment-related, local and non-adverse effects such as sinus histiocytosis in the mesenteric lymph nodes at 300 and 1000 mg/kg bw/d and granulomatous inflammation of one lung lobe with no corroborative clinical or pathological findings indicative of systemic toxicity at 100, 300 and 1000 mg/kg bw/d.