Amines, N-(3-aminopropyl)-N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01mar2002 / 30dec2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

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

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:

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

Details on test material:

- Name of test material (as cited in study report): dodecyldipropylenetriamine
- Physical state: light yellow liquid
- Analytical purity: 85.3%
-- Lot/batch No.: FP 020010155
- Expiration date of the lot/batch: 22 August 2003

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, l'Arbresle, France
- Age at study initiation: ca. 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.
- Fasting period before study:
- 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 maintance diet, batch Nos. 11217 and 20306 supplied by UAR (Villemoisson, Epinay sur Orge, France), distributed weekly, ad libitum
- Water: ad libitum
- Acclimation period: 8 days

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

Administration / exposure

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 item 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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

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, 1250 and 2500 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 2500 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 evolution and food consumption it was decided to sacrifice the animals of the highest dose-group (both males and females) for ethical reasons. At 1250 ppm food consuption 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 were 92% of controls at day 12. Those for the males show 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 1250 and 2500 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 1250 ppm group remained lower (80-90% of control), and that of the 2500 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 weeks 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.

Examinations

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 beginnin 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-day period, during the study. Food consumption was calculated per animal and per day. Achieved intake of the test item 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 beginningof the treatment period and on one occasion in week 13.
- Dose groups that were examined: before the beginning of the treatment on all aminals, 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 hr
- 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), trombocytes (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 hr
- 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 strengh, 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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

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 extremeties 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 femlaes given 300 ppm. In addition, round back (7/10 males and 10/10 females) and emaciated appearance (3/10 males and 2/10 femals) 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 scabes) 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 item 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 cntribution 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 nuber 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 item.
However, since no other disturbances of autonomic or physiological functions was observed, this effect was considered as a non-specific toxic sign and was not attributed to a direct neurotoxic effect of the test item.

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 fo 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 differet 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.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

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.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Following administration of the test substance to rats for 13 weeks by diet admixture, the NOAEL was established at 100 ppm (corresponding to 7 and 8 mg/kg/day for males and females, respectively, as active ingredient) and a LOAEL of 300 ppm (corresponding to 20 and 22 mg/kg/day for males and females, respectively, as active ingredient).
Critical effects are tubular nephropathy with associated increased ASAT activity and also Urea nitrogen levels at the highest dose.

Executive summary:

Fully compliant to OECD Guideline 408 'Subchronic Oral Toxicity - Rodent: 90-day Study' (1998).

After a 14 day range-finding using 625, 1250 and 2500 ppm Triameen Y12D, dose levels selected were: 100, 300 and 900 ppm Triameen Y12D in the diet. The control group received untreated diet. The analysis of the dietary admixtures showed satisfactory agreement between nominal and actual concentrations of the test item.

Triameen Y2D was given by dietary admixture to Sprague-Dawley rats at concentrations of 100, 300 and 900 ppm. Achieved dose of Triameen Y12D:
- Males: 7, 20 and 59 mg/kg/day
- Females: 8, 22 and 65 mg/kg bw/day

- at 100 ppm, the test item was well tolerated and there were no notable effects.

- at 300 ppm: pallor of the extremities for females. Slightly lower mean body weight gain. Higher ASAT activity. Slightly increased kidney weights. Tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes.

- at 900 ppm (for both sexes): 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. Pallor of the fundus (often associated with retinal hypovascularisation). Higher urea levels and ASAT activity, but no increase of ALAT. Pallor of the kidneys and higher weight (often associated with enlargement and higher weights) together with enlargement of the mesenteric lymph nodes. Tubular nephropathy together with foamy macrophages in the mesenteric lymph nodes. These effects on the mesenteric lymphnodes are characteristic for local irritating effect in gastro-intestinal system. Tubular nephropathy can be the result of local concentration accumulation caused by the rapid urinary excretion at a level just above cytotoxicity threshold. This is a fully reversible effect.

Consequently, following administration of DODECYLDIPROPYLENETRIAMINE to rats for 13 weeks by diet admixture, the No Observable Effect Level (NOEL) was established at 100 ppm (corresponding to 7 and 8 mg/kg/day for males and females, respectively, as active ingredient)