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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-145-8

EC Name:

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

Cas Number:

2372-82-9

Molecular formula:

C18H41N3

IUPAC Name:

bis(3-aminopropyl)(dodecyl)amine

Test material form:

liquid

Details on test material:

- Analytical purity: 89.4%
- Composition of test material, percentage of components: In a trial step, Batch no. 8-2905-1402-D drum 6 (provided by Akzo Nobel) and Batch no. DEG4918237 (provided by Lonza) were mixed at a ration of 1+1 and given the batch no. 20570-M01
- Purity test date: August 24, 2007
- Lot/batch No.: 20570-M01
- Expiration date of the lot/batch: April 30, 2009
- Storage condition of test material: At room temperature under nitrogen atmosphere in a tightly closed container, protected from heat, direct sunlight and humidity

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not 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).

Effect levels

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'

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

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).