N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 18, 2007 to April 16, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Qualifier:

according to guideline

Guideline:

other: EPA Health effects guidelines: OPPTS 870.4300 Combined chronic toxicity/carcinogenicity, August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: Males and females: 37 - 41 d
- Weight at study initiation: Males: 156.3 - 209.6 g; females 125.1 - 157.2 g
- Fasting period before study: No
- Housing: Singly
- Diet (e.g. ad libitum): Conventional laboratory diet ad libitum
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: 2 adaptation weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 15 %
- Air changes: 15 - 20 times per hr
- Photoperiod: 12/12 hrs dark / hrs light

IN-LIFE DATES: From: Apr 18, 2007 To: Apr 16, 2008

Route of administration:

oral: feed

Vehicle:

other: conventional laboratory diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance-diet mixture was freshly prepared once a week.
To maintain a constant dose level in relation to the animals’ body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex calculated for the main study animals. The concentration was adjusted weekly using the food consumption valued from two weeks earlier, e.g. the values for test week 11 for the adjustment in test week 13.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance-diet mixture was freshly prepared once a week.
- Mixing appropriate amounts with (Type of food): The appropriate amount of test substance was weighed into a glass bottle. Some of the test substance and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test substance has been distributed in the diet.
Then the premix was added to the diet, mixed with a mixer (Rhönradmischer; Type ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled according to group and dose.
- Storage temperature of food: Room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Conventional laboratory diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The method re-validation was performed as described in LPT Study Plan No. 21573. The determination of the test substance in the diet mixtures was performed applying a high performance liquid chromatography (HPLC) method with subsequent UV detection after derivatization of the test substance with 2,4-dinitro¬flourobenzene (DNFB). The concentration of the test substance in the diet mixtures was quantified applying a calibration curve calculated from the peak areas of the derivative. The results of the re-validation confirmed that the method used was reproducible and led to reliable results.The analytical method applied was based on a method given by the Sponsors and validated by LPT with regard to linearity of the calibration curve as well as accuracy, precision, stability, specificity and sensitivity.
The results of the test substance-analysis showed that the test substance-diet mixtures were correctly prepared. The results of the samples taken immediately after preparation were within 80-120 % of the nominal concentrations. However, the samples for concentration and stability (left over diet, i.e. diet that was offered to the animals for 7 d) revealed in a few instances higher concentrations as prepared (values by up to 153 %). This might have been caused by a selective feed intake by the animals.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily, oral via the diet for 52 weeks

Remarks:

Doses / Concentrations:
0, 4, 8, 20 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

100 animals (50 male and 50 female animals);
- 10 animals per sex for groups 1 (control), 2 and 3 and 20 animals per sex for group 4

Control animals:

yes, plain diet

Details on study design:

- Doses: 0, 4, 8, 20 mg/kg bw/day
- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor based on available toxicological data.
- Rationale for animal assignment (if not random): The rat is a commonly used rodent species for combined chronic toxicity/carcinogenicity studies.
- Rationale for selecting satellite groups: For part II (chronic toxicity) satellite animals were used.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Regularly throughout the working day from 7.00 a.m. to 3.45 p.m, on Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approx 3.30 p.m.
- Cage side observations checked: Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week
Once before the first administration (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals until study termination. These observations were made outside the home cage in a standard arena and at the same time, each time. Detailed records were made using scoring systems defined in the LPT SOPs. Signs noted included but were not limited to changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: At the time of group allocation, on the day of commencement of treatment and once a week thereafter, normally on the same day of the week throughout the first 13 test weeks and from test week 14 onwards throughout the experimental period in intervals of two weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the first 13 test weeks and from test week 14 onwards throughout the experimental period every second week. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of one or two treatment week(s).
- Compound intake calculated: Yes
To maintain a constant dose level in relation to the animals’ body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex calculated for the main study animals (Part I carcinogenicity). The concentration was adjusted weekly using the food consumption valued from two weeks earlier, e.g. the values for test week 11 for the adjustment in test week 13. Total and average daily intake of the test substance was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the test weeks 13, 26 and 52/53
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: 10/sex/group
- Parameters examined: Haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute), reticulocytes, platelets, haematocrit value, prothrombin time, activated partial thromboplastin time,mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of test week 4 and during the test weeks 13, 26 and 52/53
- Animals fasted: Yes (overnight)
- How many animals: 10 / sex / group
- Parameters examined: Albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), gamma-glutamyl-transferase (Gamma-GT), lactate dehydrogenase (LDH)

URINALYSIS: Yes
- Time schedule for collection of urine: During the test weeks 14, 26 and 52/53
- How many animals: 10/sex/group

- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: Volume, pH, specific gravity, protein, glucose, bilirubin, urobilinogen, ketones, Haemoglobin (Hb), nitrite,
- Microscopic examination of deposits: Epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts), crystalluria

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Test week 52
- Dose groups that were examined: 10 animals/sex/ group (in total 80 animals)
- Battery of functions tested: Sensory activity/grip strength/ motor activity

OTHER: Observational screening (righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation and auditory function.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
HISTOPATHOLOGY: Yes.

Other examinations:

Bone marrow

Statistics:

STUDENT´s t-test: Urinalysis, neurological screening
Multiple t-test based on DUNNETT, C. W.: Body weight / food consumption / haematology / clinical biochemistry / relative and absolute organ weights
Exact test of R. A. FISHER: Histopathology
Chi2 test: Bone marrow

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 specified

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
- Eight of 20 male animals treated with 20 mg/kg bw/day died during the course of the study. One in test week 31 and the other animals in test weeks 48 -53.

BODY WEIGHT AND WEIGHT GAIN:
The body weight of the rats treated with 20 mg/kg bw/day was reduced by up to 34 % from test week 15 onwards for the male animals and by up to 22% from test week 33 onwards for the female animals.
The body weight gain and body weight at autopsy changed accordingly.

FOOD CONSUMPTION AND COMPOUND INTAKE (feeding study):
The food consumption of the animals treated with 20 mg/kg bw/day was increased by up to 18% for the male rats in test weeks 39 to 45 and by up to 20% for the females in test weeks 31 to 51 caused by the severely reduced body weight.
The mean test substance intake as well as the standard deviations and minimum and maximum values are given in the following table:

Test substance intake via the diet/mean values [mg/kg bw/day]

Test substance intake via the diet/mean values [mg/kg bw/day]
Group 2 Group 3 Group 4
4 mg/kg bw/day 8 mg/kg bw/day 20 mg/kg bw/day
males females, males females, males females,
Mean 3.94, 4.00, 7.85, 8.03, 19.71, 20.02,
SD 0.31, 0.43, 0.76, 0.92, 1.56, 1.83,
Min 3.17, 3.08, 5.80, 5.81, 15.19, 16.57,
Max 4.80, 5.33, 9.47, 10.32 22.56, 23.82,


FOOD EFFICIENCY: Not examined

OPHTHALMOSCOPIC EXAMINATION : Not examined

HAEMATOLOGY:
The animals treated with 20 mg/kg bw/day revealed changes in the haemoglobin content, the numbers of leucocytes, reticulocytes, platelets, neutronphilic granulocytes, lymphocytes, monocytes, eosinophilic granulocytes, large unstained cells, basophilic granulocytes, the haematocrit value, the MCV, the MCH and the MCHC.

CLINICAL CHEMISTRY:
The animals treated with 20 mg/kg bw/day revealed increases in the plasma level of urea and in the plasma activities of ASAT and LDH in test weeks 13 to 52 as well as decreases in the plasma levels of albumin, cholesterol, glucose, protein (total) and chloride in test weeks 26 to 52.

URINALYSIS: No effects

NEUROBEHAVIOUR: No effects

ORGAN WEIGHTS:
Increased absolute kidney weights were noted for the females and decreased absolute liver weight were noted for the males treated with 20 mg/kg bw/day.

GROSS PATHOLOGY:
At necropsy, discoloured or reddened lungs were noted for the males of the high dose and enlargement of the pituitary gland was noted for the females of the intermediate and high dosed group. No histopathological correlate could be established for the pituitary.

HISTOPATHOLOGY: NON-NEOPLASTIC:
The males treated with 8 mg/kg bw/day and animals of both sexes treated with 20 mg/kg bw/day showed a dose dependent increase of lympho-histiocytic myocarditis. This finding was associated with degenerative changes of the myofibers in the heart. The males treated with 8 or 20 mg/kg bw/day and the females of the high dose group showed an increase of lympho-histiocytic infiltrations in the skeletal muscles. Degeneration of the skeletal muscles was noted in 2 intermediate dose males.
Additionally, the animals treated with 8 or 20 mg/kg bw/day showed a minimal to moderate increase in basophilic tubular cells and lympho-histiocytic infiltration of the kidney. Suppurative inflammation was observed in the prostate and mesenteric lymph nodes of the animals treated with 20 mg/kg bw/day compared to the control animals and the other test substance-treated groups. Granulomas with central neutrophilic granulocytes were only noted in the mesenteric lymph node of male and female rats of the high dose group. A high percentage of animals in all substance-treated groups without dose-related pattern had large macrophages with cytoplasmatic vacuoles in the mesenteric lymph nodes which was considered to reflect the oral route of exposure to the lipophilic test substance. Furthermore, the lungs of the male and female animals treated with 8 or 20 mg/kg bw/day showed an increased incidence of foci of foamy macrophages (alveolar histiocytosis) in the alveolus of the lungs.

HISTOPATHOLOGY: NON-NEOPLASTIC: No effects
OTHER FINDINGS:
BONE MARROW EVALUATION:
The myeloid: Erythroid ratio of the male animals treated with 20 mg/kg bw/day was significantly increased (Control: 0.993 : 1, Group 4: 1.847 : 1).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

no

Conclusions:

Under the conditions, following effects of the test substance were found: higher ASAT activity, increase of incidence of alveolar histiocytosis in the alveoli of the lungs, large macrophages in the mesenteric lymph nodes, increase of lymphohistiocytic inflammatory reactions in the kidney, skeletal muscle and heart at 8 and 20 mg a.i./kg bw/day and 40% mortality in males at the end of the year, alteration of hematological and few more clinical biochemistry parameters at 20 mg a.i./kg bw/day. The NOAEL and LOAEL were established at 4 and 8 mg a.i./kg bw/day, respectively.

Executive summary:

A combined chronic toxicity / carcinogenicity study was conducted to determine the repeated dose oral toxicity and carcinogenic effects of the test substance to Sprague Dawley rats according to the OECD Guideline 453, EU Method B.33 and US EPA OPPTS 870.4300, in compliance with GLP. In the chronic toxicity study, three groups of 10 male and 10 female rats and one group of 20 males and 20 females (for the highest dose group) were administered daily dietary levels of the test substance at 0, 100, 200, 400 ppm (corresponding to actual ingested doses of 0, 4, 8, 20 mg a.i./kg bw/day) for one year. Animals were observed for clinical signs of toxicity, body weight changes and food consumption. In addition functional battery observations, haematological, clinical chemistry, urinalysis and neuro-behavioural were performed. After Week 52, all animals were sacrificed and subjected to gross necropsy and histopathological examinations.

At 20 mg a.i./kg bw/day, the following observations were made: 40% mortality in males, reduced body weight, increased food consumption, changes in hematological parameters (such as haemoglobin content, the numbers of leucocytes, reticulocytes, platelets, neutrophilic granulocytes), altered clinical biochemistry parameters (increase in plasma level of urea, ALAT, ASAT, LDH and Gamma-GT and decrease plasma levels of albumin, cholesterol, glucose, protein (total) and chloride), discoloured or reddened lungs in males, enlargement of the pituitary gland in females (without associated histopathology), increased myeloid:erythroid ratio of the males increased absolute liver and kidney weight in males and histopathological changes (including lymphohistiocytic inflammatory reactions in the kidneys (associated with degenerative changes of the tubular epithelial cells) and skeletal muscle and heart, increased incidence of foamy macrophages in the alveoli of the lungs, inflammation mesenteric lymph nodes (including granulomas with central neutrophilic granulocytes and large macrophages with cytoplasmatic vacuoles).

At 8 mg a.i./kg bw/day, there were increase ASAT levels, enlargement of the pituitary gland in females (without associated histopathology), increased incidence of foamy macrophages in the alveoli of the lungs and large macrophages in the mesenteric lymph nodes and minimal to moderate increases of lymphohistiocytic inflammatory reactions in the kidneys of the animals.

Further, no effects on clinical signs, functional observation battery and urinalysis were noted at any of the tested dose levels.

Under the conditions, following effects of the test substance were found: higher ASAT activity, increase of incidence of alveolar histiocytosis in the alveoli of the lungs, large macrophages in the mesenteric lymph nodes, increase of lymphohistiocytic inflammatory reactions in the kidney, skeletal muscle and heart at 8 and 20 mg a.i./kg bw/day and 40% mortality in males at the end of the year, alteration of hematological and few more clinical biochemistry parameters at 20 mg a.i./kg bw/day. The NOAEL and LOAEL were established at 4 and 8 mg a.i./kg bw/day, respectively ( Leuschner, 2008).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 01, 2003 to September 13, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.27 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stefano Morini s.a.s., I-42020 S. Polo D'Enza (Reggio Emilia), Italy
- Age at study initiation: 7 months
- Weight at study initiation: Males: 9.5 -12.5 kg; females: 8.0 - 11.6 kg
- Diet (e.g. ad libitum): Powder feed (ssniff Hd-D V3231), 40 g/kg bw daily.
- Water: Ad libitum
- Housing: Singly or by twos in kennels. Each kennel had an inside yard and outside yard of total 9 m2. These cages are specially equipped to keep the animals singly during the daily examinations. The outside yard was accessible at all times (except at the time of feeding and examinations).
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): The test substance and powder feed (ssniff® Hd-H V3231) were mixed with a mixer (Rohn-Radmischer; J. Engelmann, Ludwigshafen, Germany).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC-UV/VIS

Duration of treatment / exposure:

90 d

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 (Group 1), 200 ppm (Group 2), 500 ppm (Group 3), 1,500 ppm (Group 4).
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 8.0, 20.0, 55.1/52.6 mg kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

4/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of the dose range finding study. Administration of the fixed doses of 200 and 500 ppm test substance for 28 d resulted in no signs of toxicity. A dose level of 1,500 ppm caused diarrhoea and reduced food intake (in the females). Body weight, heart rate, circulatory functions, haematological and biochemical parameters and the relative or absolute weights of kidney were not influenced by the treatment with 200, 500 and 1,500 ppm during the fixed dose period. At necropsy a red discoloured lymph node (cervical, mesenteric and/or pulmonal) and reddened and/or thickened mucosa of the gastro-intestinal tract were observed in nearly all animals treated with 200, 500 and 1,500 ppm during the fixed dose period. An infiltration of foamy macrophages was noted in the mucosa of the jejunum and/or ileum and in the mesenteric lymph nodes of the 500 and 1,500 ppm dose group.
- Rationale for animal assignment (if not random): To obtain approximately equal mean body weights in all groups

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked regularly throughout the working day from 7.00 a.m. to 4.00 p.m. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m. Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals.
- Kennelside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of all animals was recorded at study initiation (first administration of test substance) and thereafter in weekly intervals always on the same weekday.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a daily basis throughout the experimental period. From these data the quantities consumed were calculated. Any uneaten food was removed, the residue recorded and discarded. The report included weekly mean values.
From these data the relative food consumption (in g/kg b.w./day) was determined.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption of all animals was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the first administration and at the end of the 90-d treatment period.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the first administration, at the end of test weeks 6 and 13
- Animals fasted: Yes, overnight
- How many animals: All 36 animals of the pool before the first administration and 32 selected animals of the pool at the end of test weeks 6 and 13
- Parameters examined: Differential blood count, erythrocyte count, leucocyte count, haematocrit value, haemoglobin content, platelets, reticulocytes, throm-boplastin time, activated partial thromboplastin time, MCV, MCH and MCHC

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before the first administration, at the end of test weeks 6 and 13
- Animals fasted: Yes, overnight
- How many animals: All 36 animals of the pool before the first administration and 32 selected animals of the pool at the end of test weeks 6 and 13
- Parameters examined: Albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, inorganic phosphate, protein (total), triglycerides, urea (in blood), chloride, potassium, sodium, calcium, ALAT, aP, ASAT, Gamma-GT.

URINALYSIS: Yes
- Time schedule for collection of urine: before the first administration for all 36 animals pool and at the end of test weeks 6 and 13 (32 animals selected the pool).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters examined: Volume, colour, specific gravity, pH, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin, nitrite. Microscopic examinations of urine samples: the deposits were examined for the presence of the following parameters: epithelial cells, leucocytes, erythrocytes, organisms, further constituents (e.g. sperm, casts), crystalluria.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
- Organs examined: Adrenals, aorta abdominalis, bone (sternum and osfemoris with joint), bone marrow (sternum and femur), brain (cerebrum, cerebellum, medulla/pons), caecum, epididymis, eye with optic nerve, gall bladder, gross lesions observed, heart (left and right ventricle, septum), intestines (small and large), kidneys and ureter, lacrimal glands, liver, lungs (with mainstem broni and bronchioles), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands (mandibular, parotid, sublingual), seminal duct, skin (left flank), spinal cord (cervical, thoracic, lumbar), spleen, stomach, testicles, thymus, thyroids (incl. parathyroids), tissue masses or tumours, trachea (incl. larynx), urinary bladder, uterus (md. cervix and oviducts), vagina.

HISTOPATHOLOGY: Yes

Other examinations:

Organ weights were determined for the following organs: Adrenals, brain, epididymis, gall bladder, heart, kidneys, liver, ovaries, spleen, testicles, thymus, thyroids (incl. parathyroids), uterus.
The auditory acuity was checked with a simple noise test.

Statistics:

Statistical analysis of data was performed for male and female animals separately. Group mean values with standard deviation were calculated for all parameters with exception of differential counts, for which median and range were determined. For body weights, food consumption, haematology, clinical biochemistry and relative organ weights: Multiple t-test based on Dunnett (p= 0.01), the following limit was used: p = 0.01 . t = 3.58 for 12 degrees of freedom; for urinalysis: Student-s t-test (p = 0.01) and for histopathology: Exact test of R.A. Fisher (p= 0.05)

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
None of the dogs treated with 200 or 500 ppm showed any clinical signs of systemic toxicity. Emesis was observed in two high-dosed male animals on test Days 2 or 6. The effect started 2-6 h after administration and lasted up to 5 min. The emesis was considered to be within the normal variability and not test substance-related. The high-dosed female animals did not show any clinical signs of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
No effect on the body weight was noted at dose levels of 200 or 500 ppm. Compared to the control animals the body weight of the high dosed animals (1,500 ppm) was reduced up to 18% in the males and up to 12% in the females during the treatment period, although statistical significance was not reached.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No influence was observed on the food consumption of the male and female animals treated with 200 or 500 pp, while statistically significant decrease was observed in the high dose group animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Drinking water consumption, monitored by visual appraisal, did not show any test substance-related influence.

OPHTHALMOSCOPIC EXAMINATION
Eyes and hearing were not influenced.

HAEMATOLOGY
No test substance-related influence was noted on the haematological parameters.

CLINICAL CHEMISTRY
Treatment with 200 or 500 ppm did not influence the clinical biochemistry parameters examined in test week 6. Treatment with 1,500 ppm caused in increase in plasma ASAT activity in both sexes (statistically significant in males). In test week 13 the statistically significant increases in ASAT and ALAT plasma activity were noted for the male and female animals treated with 500 or 1,500 ppm when compared with the control group. An increased level of potassium was also observed at the high dosed animals.

URINALYSIS
No test substance-related changes were noted for the specific gravity, the pH-value or the urinary status.

ORGAN WEIGHTS
No test substance-related changes were noted for the relative and absolute organ weights of the animals treated with 200 or 500 ppm. Increased relative and absolute weights of the gall bladder were noted in animals of both sexes of the high dose group. The deviation in males was slight and did not reach statistical significance.

GROSS PATHOLOGY
No test substance-related changes were noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histomorphological examination did not reveal morphological systemic changes which are considered to be related to the administration of the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on increased ALAT and ASAT values at 500 ppm (i.e., 20 mg a.i./kg bw/day)

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the NOEL was established at 200 ppm (8 mg a.i./kg bw/day) due to increased ALAT and ASAT values observed at 500 ppm (20 mg a.i./kg bw/day). However, due to the absence of pathological changes and any other signs of systemic toxicity, these changes at 500 ppm (20 mg a.i./kg bw/day) were considered to be non-adverse and the NOAEL was established at 500 ppm (20 mg a.i./kg bw/day).

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance in Beagle dogs according to OECD Guideline 409, US EPA OPPTS 870.3150 and EU Method B.27, in compliance with GLP. Groups of 4 male and 4 female dogs were administered daily dietary levels of 0, 200, 500 and 1500 ppm (corresponding to actual ingested doses of 0, 8.0, 20.0, 55.1 (males) and 52.6 (females) mg a.i./kg bw/day) for 90 days. Animals were observed for clinical signs of toxicity, body weight changes and food consumption. In addition functional battery observations and haematological, clinical chemistry, ophthalmological examinations and urinalysis were performed. All animals were sacrificed and subjected to gross necropsy and histopathological examinations. Treatment with either 500 or 1500 ppm resulted in dose-related in- creased plasma activity of ALAT and ASAT. The high dose of 1500 ppm caused reduced food consumption, reduced body weight and increased relative and absolute weights of the gall bladder. An increased plasma level of potassium was also observed at the high dose level. No test substance-related influence was noted on the haematological parameters, the eyes or optic region, the auditory acuity and the urinary status at any of the dose levels. None of the animals died prematurely. At necropsy no test substance-related changes were observed in the organs/tissues of the dogs. The histopathological examination did not reveal any test substance-related changes. Under the study conditions, the NOEL was established at 200 ppm (8 mg a.i./kg bw/day) due to increased ALAT and ASAT values observed at 500 ppm (20 mg a.i./kg bw/day). However, due to the absence of pathological changes and any other signs of systemic toxicity, these changes at 500 ppm (20 mg a.i./kg bw/day) were considered to be non-adverse and the NOAEL was established at 500 ppm (20 mg a.i./kg bw/day) (Leuschner, 2004).

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days, 4 week post-exposure

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 1.5, 3, 9, 27 mg active ingredient/kg bw

No. of animals per sex per dose:

10 per sex per dose level
A further group of 5 animals/sex was used for baseline blood analyses.

Control animals:

yes, concurrent vehicle

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No nephrotoxic effects were seen in either sex at 30 mg/kg bw/day (9 mg a.i./kg bw/day). Nephrotoxicity was the only finding considered to represent systemic toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No nephrotoxic effects were seen in either sex at this dose. Nephrotoxicity was the only finding considered to represent systemic toxicity.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Conclusions:

Under the study conditions, the rat 90 d NO(A)EL was established at 9 mg a.i./kg bw/day.

Executive summary:

A study was conducted to determine repeated dose toxicity of the test substance in rats according to OECD Guideline 408, in compliance with GLP. The test substance was administrated (in water) to Wistar rats (male/female) by oral gavage for 90 d. The post-exposure period lasted 4 weeks. Ten animals per group were treated at doses level of 0, 1.5, 3, 9 and 27 mg a.i./kg bw. A further group of 5 animals/sex was used for baseline blood analyses. No nephrotoxic effects were seen in either sex at 9 mg a.i./kg bw/day. Nephrotoxicity was the only finding considered to represent systemic toxicity. Under the study conditions, the rat 90 d NO(A)EL was established at 9 mg a.i./kg bw/day (Korn, 1992; Prentice, 1999).

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 01, 2002 to December 30, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Cited as Directive 88/302/EEC, B.26

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: Approx. 6 weeks old
- Weight at study initiation: Mean 206 g (range: 194 g to 221 g) for the males and 176 g (range: 163 g to 201 g) for the females.
- Housing: Suspended wire mesh cages (43.0 x 21.5 x 18 cm), 2 rats of the same sex and group
- Diet: A04C P.25 treated or untreated powdered maintainance diet, batch Nos. 11217 and 20306 supplied by UAR (Villemoisson, Epinay sur Orge, France), distributed weekly, ad libitum
- Water: Ad libitum
- Acclimation period: 8 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): The test substance was mixed with a small quantity of diet using a mortar and a pestle. Then, this premix was blended with the remaining diet in a Lodige FM50 (ATR, France) for a period of 10 minutes in order to obtain the concentrations of 100, 300 and 900 part per million (ppm).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13-week

Frequency of treatment:

Daily in feed

Remarks:

Doses / Concentrations:
0, 100, 300, 900 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
males: 7, 20 and 57 mg/kg bw/day; females: 8, 22 and 65 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE:
Dose levels were chosen based on the results of preliminary 2-week dose range finding study using 0, 625, 1,250 and 2,500 ppm in the diet, using 6 males and 6 females per dose group. This corresponded to the ingested doses of 57, 103 and 126 mg/kg bw/day for males and 61, 107 and 178 mg/kg bw/day for males and females, respectively.
- At 2,500 ppm dose level the palatability was so bad that the animals suffered from malnutrition. Two male animals of this group were found dead on Day 12. Following these results and those concerning the body weight evaluation and food consumption it was decided to sacrifice the animals of the highest dose-group (both males and females) for ethical reasons.
- At 1,250 ppm food consumption was (just) acceptable, reaching 83 % in males and 94 % in females of the controls. In both groups body weight dropped to about 80 % of the controls at Day 12.
- In 625 ppm group food consumption at Day 5 (per kg bw) was about the same as the control (97 % males, 107% females). The body weight for the females was 92 % of controls at Day 12.
- Those for the males showed a strange pattern: a sudden drop in body weight between Day 8 and 12 was observed in 2 animals (287 g on Day 8, 228 g Day 12 and 314 g Day 8, 251 g Day 12), while until Day 8 there was a steady body weight gain. There was an initial drop in the body weight at the start of the study, especially at the 1,250 and 2,500 ppm groups. Following the information on Day 5, it seems that the animals in the low-dose group have become accustomed to it, while that in the 1,250 ppm group remained lower (80-90 % of control), and that of the 2,500 ppm group recovered somewhat, but still remained unacceptable. None of the animals in the two lower dose groups showed clinical effects.
Based on these results the following doses were chosen for the 13-week study: 100, 300 and 900 ppm.

RATIONALE FOR ANIMAL ASSIGNMENT (if not random): The animals were assigned to the test groups so that the average body weight of each group was similar.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice a day, except weekends and public holidays
- Cage side observations: Mortality or signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One before the beginning of the treatment period and one a week thereafter.
Observations included (but were not limited to) changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Once before allocation of the animals to groups, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

The quantity of food consumed by the animals of each cage was recorded once a week, over a 7-d period, during the study. Food consumption was calculated per animal and per d. Achieved intake of the test substance was calculated on a weekly basis for each treated group as follows: D = C*(FC/BW), where D = achieved dosage (mg/kg/day), C = nominal concentration (ppm), FC = mean food consumption (g/animal/day) and BW = mean body weight (g).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the beginning of the treatment period and on one occasion in week 13.
- Dose groups that were examined: Before the beginning of the treatment on all animals, in week 13 on animals of the control and high-dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight for at least 14 h
- How many animals: All animals
- Parameters examined: Erythrocytes (RBC), hemoglobin (HB), mean cell volume (MCV), packed cell volume (PCV), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLAT), leukocytes (WBC), differential white cell count with cell morphology, neutrophils (N), eosinophils (E), basophils (B), lymphocytes (L), monocytes (M) and prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Animals fasted: Yes, overnight for at least 14 h
- How many animals: All animals
- Parameters examined: Sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca2+), inorganic phosphorus (I.PHOS), glucose (GLUC), urea (UREA), creatinine (CREAT), total bilirubin (TOT.BIL), total protein (PROT), albumin (ALB), albumin/globulin ratio (A/G), cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once at the end of the treatment period
- Dose groups that were examined: All animals
- Battery of functions tested: Grip strength, measurement of reactivity to manipulation or to different stimuli and motor activity. The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
- in the hand: Fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): Grooming, palpebral closure, defecation, and urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior and breathing, ataxia and hypotonia.
Then, the following parameters measurements, reflexes and responses were recorded:
-touch response,
-forelimb grip strength,
-papillary reflex,
-visual stimulus response,
-auditory startle reflex,
-tail pinch response,
-righting reflex,
-landing foot splay,
-at the end of observation: Rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 15-minute period at the end of the treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The following organs were examined:
- Adrenals;
- Aorta;
- Brain (including medulla/pons; cerebellar and cerebral corte);
- Cecum;
- Colon;
- Duodenum;
- Epididymides;
- Esophagus;
- Heart;
- Ileum;
- Jejunum;
- Kidneys;
- Liver;
- Lungs with bronchi;
- Lymph nodes (mandibular and mesenteric);
- Mammary glands/area;
- Ovaries (with oviducts);
- Pancreas;
- Pituitary gland;
- Prostate (dorso-lateral and ventral);
- Rectum;
- Salivary glands (sublingual and submandibular);
- Sciatic nerve;
- Seminal vesicles (including coagulation gland);
- Skin;
- Spinal cord (cervical, thoracic and lumbar);
- Spleen;
- Sternum with bone marrow;
- Stomach with forestomach;
- Testes;
- Thymus;
- Thyroids with parathyroids;
- Trachea;
- Urinary bladder;
- Uterus (horns and cervi);
- Vagina.
(Preservation of tissue: Eyes with Harderian glands; Femoral bone with articulation; Tongue; Skeletal muscle.)

HISTOPATHOLOGY: Yes
The following tissues were examined: all tissues for animals of the control and high-dose groups (groups 1 and 4), kidneys, mesenteric lymph nodes and all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3).

Statistics:

Depending on normal distribution (Kolmogorov-Lilliefors test, possibly after a logarithmic transformation of the values - except for organ weights) and following testing of homogeneity of variances between groups: Bartlett test (3 or more groups) or Fisher test (2 groups): If Homogeneous Student test (2 groups) Dunnett test (3 or more groups), else Dunn test (3 or more groups) Mann-Whitney/Wilcoxon test (2 groups) If not normal distribution: Dunn test

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
Pallor of the extremities and piloerection were noted in all males and females given 900 ppm, starting generally from weeks 5 or 7 of the study. Pallor of the extremities was also seen in all females given 300 ppm. In addition, round back (7/10 males and 10/10 females) and emaciated appearance (3/10 males and 2/10 females) were observed in animals given 900 ppm from the second half of the study period.
From week 11 until the end of the study chromodacryorrhea was noted for 2/10 females given 900 ppm and 1/10 females given 300 ppm.
Hair loss (sometimes associated with the presence of scabs) on the right and/or left forelimbs, the head, the neck and/or the back, was observed in a few control and test-treated animals at all dose levels. Swelling of the ears was seen in one male given 100 ppm, generally towards the end of the study period.
In view of their high incidence, the pallor of the extremities (at 300 ppm in females and 900 ppm in both sexes), piloerection and round back (both at 900 ppm in all animals) were considered to be test substance related and of toxicological relevance. All other clinical signs were considered to be of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN:
When compared to controls, a markedly lower mean body weight gain was noted in both sexes at 900 ppm (-46 % in males and -51% in females, statistically significant) from week 3 onwards. A lower body weight gain was also observed for males given 300 ppm (-10 %_. This effect increased with time and was severe from week 8 onwards.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the study, the daily food consumption was similar for all animals groups with the exception of the males and females given 900 ppm, which showed a moderately lower mean daily food consumption (statistically significant, weeks 4 to 14) over almost the whole study period in comparison to the controls. This effects was considered to be related to the test treatment and contributed to the lower body weight gains noted for these animals over weeks 4 to 14 of the study period.

OPHTHALMOSCOPIC EXAMINATION:
Pallor of the fundus, which was often associated with retinal hypocascularization, was noted with a high incidence in both males and females given 900 ppm (5/10 test-treated males vs. 0/10 control males, 5/10 test treated females vs. 1/10 control females) at the end of the study period. In view of the incidence of this ophthalmological finding for animals given the top dose-level, a relationship to the treatment cannot be excluded.
The few other ophthalmological findings were those which are commonly recorded spontaneously in the untreated laboratory rats of this strain and age and were considered to be of no toxicological importance.

HAEMATOLOGY:
The differences in the red blood cell parameters were minimal, the contribution of few individual animals, and the individual values were within the range of the historical background data of the test lab. Therefore they were considered of no toxicological importance.
Similarly, the differences in platelet count and the other parameters of coagulation were sometimes not dose-related and were the contribution of low values in some control animals and high values occasionally observed in the test animals. Therefore they were considered of no toxicological importance.
Taking into account the great variability of the total and differential white blood cell count and that the individual values were within or near the range of the historical background data of the test lab, they were also considered of no toxicological importance.

CLINICAL CHEMISTRY:
The higher urea nitrogen level in the males and females given 900 ppm and ASAT activity in the males and females given 300 ppm and 900 ppm were considered to be treatment-related.
As all other differences were minimal, sometimes not dose-related and were the contribution of high values in some control animals, they were considered to be of no toxicological importance.

NEUROBEHAVIOUR:
A slight decrease in motor activity (total number of movements) was observed in males and females given 900 ppm. As seen only at the high dose-level, this finding was considered to be related to the test substance.
However, since no other disturbances of autonomic or physiological functions were observed, this effect was considered as a non-specific toxic sign and was not attributed to a direct neurotoxic effect of the test substance.

ORGAN WEIGHTS:
Dose-related higher kidney weight was observed for animals of both sexes given 300 ppm and 900 ppm. This was considered to be treatment-related and correlated with the tubular nephropathy observed among these animals.
As the differences in the spleen and thymus weights were the contribution for very few individual animals and were without relevant histopathological abnormalities, they were considered to be of no toxicological importance.
Other differences were considered to be of no toxicological importance, as they were minimal, sometimes of opposing trend in the two sexes and different groups and were not accompanied by relevant histopathological findings.

GROSS PATHOLOGY:
Pallor of kidneys was seen in 1/10 females given 300 ppm and in five males and all females given 900 ppm (often associated here with enlargement). Enlargement of the mesenteric lymph nodes was noted among the animals of both sexes given 300 and 900 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Tubular nephropathy (characterized by tubular epithelial cell degeneration/necrosis, tubular dilatation with flattened epithelium and tubular basophilia) of the kidneys was noted in animals given 300 and 900 ppm (with variable severity) and correlated with the macroscopic findings. Foamy macrophages were noted in the mesenteric lymph nodes of all males and females given 300 ppm and in 9 males and all females given 900 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 - <= 8 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on tubular nephropathy of the kidney, associated with increased relative kidney weight, and foamy macrophages noted in the mesenteric lymphnodes at the higher dose levels.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the study conditions, the following effects of the test substance were found: tubular nephropathy of the kidney, associated with increased relative kidney weight, and foamy macrophages noted in the mesenteric lymph nodes at the higher dose levels. The LOAEL and NOAEL were established at 300 and 100 ppm (equivalent to 20 and 7 mg a.i./kg/day for males and 22 and 8 mg a.i./kg bw/day for females), respectively.

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the test substance in Sprague Dawley rats according to EU Method B.26, in compliance with GLP. Groups of 10 male and 10 female rats were administered daily dietary levels of 0, 100, 300 and 900 ppm (corresponding to actual ingested doses of 7, 20 and 57 mg a.i./kg bw/day in males and 8, 22 and 65 mg a.i./kg bw/day in females) for 90 d. Animals were observed for clinical signs of toxicity, bodyweight changes and food consumption. In addition, functional battery observations and haematological, clinical chemistry and ophthalmological examinations were performed. All animals were sacrificed and subjected to gross necropsy and histopathological examinations. At 100 ppm, the test substance was well tolerated and there were no notable effects. At 300 ppm, observed effects included pallor of the extremities for females, slightly lower mean body weight gain, higher ASAT activity, slightly increased kidney weights and tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes. At 900 ppm (for both sexes), there were clinical signs (such as pallor of the extremities, piloerection, round back and slight decrease in motor activity), markedly lower mean body weight gain together with a markedly lower food consumption, biochemical changes (including higher urea levels and ASAT activity, but no increase of ALAT), pallor of the fundus (often associated with retinal hypovascularisation), pallor of the kidneys and higher weight (often associated with enlargement and higher weights) together with enlargement of the mesenteric lymph node and, tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes. These effects on the mesenteric lymph nodes are characteristic of local irritation in the gastro-intestinal system. Tubular nephropathy could have been be the result of local accumulation caused by the rapid urinary excretion at a level just above the cytotoxicity threshold. This is fully reversible. Under the study conditions, the following effects of the test substance were found: tubular nephropathy of the kidney, associated with increased relative kidney weight, and foamy macrophages noted in the mesenteric lymph nodes at the higher dose levels. The LOAEL and NOAEL were established at 300 and 100 ppm (equivalent to 20 and 7 mg a.i./kg/day for males and 22 and 8 mg a.i./kg bw/day for females), respectively (Mhedhbi, 2003).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

yes

Remarks:

see, principles of method if other than guideline

Principles of method if other than guideline:

The high dose level was lowered because of the severe irritation (eschar formation) of the application skin site as it could potentially decrease dermal absorption of the test substance. This change was not considered to effect the integrity of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 hours/day, 90 days

Frequency of treatment:

5 days a week

Remarks:

Doses / Concentrations:
0.25%, 0.5% and 1.0% a.i. (1.0% reduced to 0.75% after approximately one week of dosing), corresponding to 5, 10 and 20 (15) mg a.i./kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

Cohort I : 46 rats/sex,
5 dose groups of 8 rats/sex/group
Cohort II: 69 rats/sex
4 groups of 15 rats/sex/group

Control animals:

yes

Key result

Dose descriptor:

NOEL

Remarks:

(systemic effects)

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no effects at any tested dose

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Critical effects observed:

no

NOEL was established at 15 mg a.i./kg bw. No evidence of an immunotoxicity or systemic toxicity was seen following dermal application of the test substance at dose levels up to 15 mg/kg bw for 6-7 (immuntoxicity evaluation) or 13-14 (systemic toxicity) weeks. All effects seen in the study were attributed to local irritation caused by dermal application of the test substance. With respect to local skin effects, no NOAEL was established.

Conclusions:

Under the study conditions, the 90 d NOEL of the test substance was established at 15 mg a.i./kg bw in rats.

Executive summary:

A study was conducted to determine dermal repeated dose toxicity of the test substance to Sprague Dawley rats for a minimum of 90 d according to EPA OPP Guideline 82-3, in compliance with GLP. Animals were exposed to the test substance at concentrations of 5, 10 and 20 (15) mg a.i./kg bw/day for duration of 6 h/day. There were no evidence of immunotoxicity or systemic toxicity following dermal application of the test substance at dose levels up to 15 mg a.i./kg bw/day for 6-7 (immunotoxicity evaluation) or 13-14 (systemic toxicity) weeks. All effects seen in the study were attributed to local irritation. With respect to local skin effects, no NOAEL was established. Under the study conditions, the 90 d NOEL of the test substance was established at 15 mg a.i./kg bw in rats (Johnson, 2000).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

yes

Remarks:

see, principles of method if other than guideline

Principles of method if other than guideline:

The high dose level was lowered because of the severe irritation (eschar formation) of the application skin site as it could potentially decrease dermal absorption of the test substance. This change was not considered to effect the integrity of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 hours/day, 90 days

Frequency of treatment:

5 days a week

Remarks:

Doses / Concentrations:
0.25%, 0.5% and 1.0% a.i. (1.0% reduced to 0.75% after approximately one week of dosing), corresponding to 5, 10 and 20 (15) mg a.i./kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

Cohort I : 46 rats/sex,
5 dose groups of 8 rats/sex/group
Cohort II: 69 rats/sex
4 groups of 15 rats/sex/group

Control animals:

yes

Key result

Dose descriptor:

NOEL

Remarks:

(systemic effects)

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no effects at any tested dose

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Critical effects observed:

no

NOEL was established at 15 mg a.i./kg bw. No evidence of an immunotoxicity or systemic toxicity was seen following dermal application of the test substance at dose levels up to 15 mg/kg bw for 6-7 (immuntoxicity evaluation) or 13-14 (systemic toxicity) weeks. All effects seen in the study were attributed to local irritation caused by dermal application of the test substance. With respect to local skin effects, no NOAEL was established.

Conclusions:

Under the study conditions, the 90 d NOEL of the test substance was established at 15 mg a.i./kg bw in rats.

Executive summary:

A study was conducted to determine dermal repeated dose toxicity of the test substance to Sprague Dawley rats for a minimum of 90 d according to EPA OPP Guideline 82-3, in compliance with GLP. Animals were exposed to the test substance at concentrations of 5, 10 and 20 (15) mg a.i./kg bw/day for duration of 6 h/day. There were no evidence of immunotoxicity or systemic toxicity following dermal application of the test substance at dose levels up to 15 mg a.i./kg bw/day for 6-7 (immunotoxicity evaluation) or 13-14 (systemic toxicity) weeks. All effects seen in the study were attributed to local irritation. With respect to local skin effects, no NOAEL was established. Under the study conditions, the 90 d NOEL of the test substance was established at 15 mg a.i./kg bw in rats (Johnson, 2000).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Mode of Action Analysis / Human Relevance Framework

The most significant treatment-related changes in all studies performed on polyamines such as the present test substance are effects on the small intestine and mesenteric lymph nodes. A relatively strong inflammatory reaction is also observed at high dose levels. These effects in the gastro-intestinal tract have consistently been observed with these polyamines.

A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. The observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and generally considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance, and for substances belonging to the same group of chemicals, and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect.

Phospholipidosis is a plausible mechanism. In physiological circumstances, the triamine has a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tail easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil. The complex of cationic surfactant and phospholipids are difficult to digest by the macrophages, and they accumulate with the lysosomes. Recent (unpublished) studies have shown that these cationic surfactants are all lysosomotropic, and scored positive for phospholipidosis in in vitro studies with HepG2 cells.

Additional information

Oral

90 d studies (rat and dog)

A study was conducted to determine the repeated dose oral toxicity of the test substance in Sprague Dawley rats according to EU Method B.26, in compliance with GLP. Groups of 10 male and 10 female rats were administered daily dietary levels of 0, 100, 300 and 900 ppm (corresponding to actual ingested doses of 7, 20 and 57 mg a.i./kg bw/day in males and 8, 22 and 65 mg a.i./kg bw/day in females) for 90 d. Animals were observed for clinical signs of toxicity, bodyweight changes and food consumption. In addition, functional battery observations and haematological, clinical chemistry and ophthalmological examinations were performed. All animals were sacrificed and subjected to gross necropsy and histopathological examinations. At 100 ppm, the test substance was well tolerated and there were no notable effects. At 300 ppm, observed effects included pallor of the extremities for females, slightly lower mean body weight gain, higher ASAT activity, slightly increased kidney weights and tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes. At 900 ppm (for both sexes), there were clinical signs (such as pallor of the extremities, piloerection, round back and slight decrease in motor activity), markedly lower mean body weight gain together with a markedly lower food consumption, biochemical changes (including higher urea levels and ASAT activity, but no increase of ALAT), pallor of the fundus (often associated with retinal hypovascularisation), pallor of the kidneys and higher weight (often associated with enlargement and higher weights) together with enlargement of the mesenteric lymph node and, tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes. These effects on the mesenteric lymph nodes are characteristic of local irritation in the gastro-intestinal system. Tubular nephropathy could have been be the result of local accumulation caused by the rapid urinary excretion at a level just above the cytotoxicity threshold. This is fully reversible. Under the study conditions, the following effects of the test substance were found: tubular nephropathy of the kidney, associated with increased relative kidney weight, and foamy macrophages noted in the mesenteric lymph nodes at the higher dose levels. The LOAEL and NOAEL were established at 300 and 100 ppm (equivalent to 20 and 7 mg a.i./kg/day for males and 22 and 8 mg a.i./kg bw/day for females), respectively (Mhedhbi, 2003).

A study was conducted to determine repeated dose toxicity of the test substance in rats according to OECD Guideline 408, in compliance with GLP. The test substance was administrated (in water) to Wistar rats (male/female) by oral gavage for 90 d. The post-exposure period lasted 4 weeks. Ten animals per group were treated at doses level of 0, 1.5, 3, 9 and 27 mg a.i./kg bw. A further group of 5 animals/sex was used for baseline blood analyses. No nephrotoxic effects were seen in either sex at 9 mg a.i./kg bw/day. Nephrotoxicity was the only finding considered to represent systemic toxicity. Under the study conditions, the rat 90 d NO(A)EL was established at 9 mg a.i./kg bw/day (Korn, 1992; Prentice, 1999).

A study was conducted to determine the repeated dose oral toxicity of the test substance in Beagle dogs according to OECD Guideline 409, US EPA OPPTS 870.3150 and EU Method B.27, in compliance with GLP. Groups of 4 male and 4 female dogs were administered daily dietary levels of 0, 200, 500 and 1500 ppm (corresponding to actual ingested doses of 0, 8.0, 20.0, 55.1 (males) and 52.6 (females) mg a.i./kg bw/day) for 90 days. Animals were observed for clinical signs of toxicity, body weight changes and food consumption. In addition functional battery observations and haematological, clinical chemistry, ophthalmological examinations and urinalysis were performed. All animals were sacrificed and subjected to gross necropsy and histopathological examinations. Treatment with either 500 or 1500 ppm resulted in dose-related in- creased plasma activity of ALAT and ASAT. The high dose of 1500 ppm caused reduced food consumption, reduced body weight and increased relative and absolute weights of the gall bladder. An increased plasma level of potassium was also observed at the high dose level. No test substance-related influence was noted on the haematological parameters, the eyes or optic region, the auditory acuity and the urinary status at any of the dose levels. None of the animals died prematurely. At necropsy no test substance-related changes were observed in the organs/tissues of the dogs. The histopathological examination did not reveal any test substance-related changes. Under the study conditions, the NOEL was established at 200 ppm (8 mg a.i./kg bw/day) due to increased ALAT and ASAT values observed at 500 ppm (20 mg a.i./kg bw/day). However, due to the absence of pathological changes and any other signs of systemic toxicity, these changes at 500 ppm (20 mg a.i./kg bw/day) were considered to be non-adverse and the NOAEL was established at 500 ppm (20 mg a.i./kg bw/day) (Leuschner, 2004).

1 year study (rat)

A combined chronic toxicity / carcinogenicity study was conducted to determine the repeated dose oral toxicity and carcinogenic effects of the test substance to Sprague Dawley rats according to the OECD Guideline 453, EU Method B.33 and US EPA OPPTS 870.4300, in compliance with GLP. In the chronic toxicity study, three groups of 10 male and 10 female rats and one group of 20 males and 20 females (for the highest dose group) were administered daily dietary levels of the test substance at 0, 100, 200, 400 ppm (corresponding to actual ingested doses of 0, 4, 8, 20 mg a.i./kg bw/day) for one year. Animals were observed for clinical signs of toxicity, body weight changes and food consumption. In addition functional battery observations, haematological, clinical chemistry, urinalysis and neuro-behavioural were performed. After Week 52, all animals were sacrificed and subjected to gross necropsy and histopathological examinations.

At 20 mg a.i./kg bw/day, the following observations were made: 40% mortality in males, reduced body weight, increased food consumption, changes in hematological parameters (such as haemoglobin content, the numbers of leucocytes, reticulocytes, platelets, neutrophilic granulocytes), altered clinical biochemistry parameters (increase in plasma level of urea, ALAT, ASAT, LDH and Gamma-GT and decrease plasma levels of albumin, cholesterol, glucose, protein (total) and chloride), discoloured or reddened lungs in males, enlargement of the pituitary gland in females (without associated histopathology), increased myeloid:erythroid ratio of the males increased absolute liver and kidney weight in males and histopathological changes (including lymphohistiocytic inflammatory reactions in the kidneys (associated with degenerative changes of the tubular epithelial cells) and skeletal muscle and heart, increased incidence of foamy macrophages in the alveoli of the lungs, inflammation mesenteric lymph nodes (including granulomas with central neutrophilic granulocytes and large macrophages with cytoplasmatic vacuoles).

At 8 mg a.i./kg bw/day, there were increase ASAT levels, enlargement of the pituitary gland in females (without associated histopathology), increased incidence of foamy macrophages in the alveoli of the lungs and large macrophages in the mesenteric lymph nodes and minimal to moderate increases of lymphohistiocytic inflammatory reactions in the kidneys of the animals.

Further, no effects on clinical signs, functional observation battery and urinalysis were noted at any of the tested dose levels.

Under the conditions, following effects of the test substance were found: higher ASAT activity, increase of incidence of alveolar histiocytosis in the alveoli of the lungs, large macrophages in the mesenteric lymph nodes, increase of lymphohistiocytic inflammatory reactions in the kidney, skeletal muscle and heart at 8 and 20 mg a.i./kg bw/day and 40% mortality in males at the end of the year, alteration of hematological and few more clinical biochemistry parameters at 20 mg a.i./kg bw/day. The NOAEL and LOAEL were established at 4 and 8 mg a.i./kg bw/day, respectively ( Leuschner, 2008).

Dermal

A study was conducted to determine dermal repeated dose toxicity of the test substance to Sprague Dawley rats for a minimum of 90 d according to EPA OPP Guideline 82-3, in compliance with GLP. Animals were exposed to the test substance at concentrations of 5, 10 and 20 (15) mg a.i./kg bw/day for duration of 6 h/day. There were no evidence of immunotoxicity or systemic toxicity following dermal application of the test substance at dose levels up to 15 mg a.i./kg bw/day for 6-7 (immunotoxicity evaluation) or 13-14 (systemic toxicity) weeks. All effects seen in the study were attributed to local irritation. With respect to local skin effects, no NOAEL was established. Under the study conditions, the 90 d NOEL of the test substance was established at 15 mg a.i./kg bw in rats (Johnson, 2000).

Justification for classification or non-classification

Based on the results of the repeated dose oral toxicity studies with rats showing kidney pathology (tubular nephropathy of the kidneys) at levels between 10-100 mg/kg bw/day (average of 21 mg/kg bw/day), the substance warrants classification as STOT RE 2 - H373 (May cause damage to organs (kidney) through prolonged or repeated oral exposure) according to CLP (EC 1272/2008) criteria.