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

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2007 - April 2008 (chronic toxicity) April 2009 (carcinogenicity)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain / Stock: CD® / Crl:CD(SD); Charles River Laboratories Germany GmbH
At start of treatment:
Age: 37 - 41 days;
body weight: Males: 156.3 - 209.6 g; Females: 125.1 - 157.2 g

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Food consumption per day ad libitum. Concentrations in food adjusted to reach test substance intake of: 4, 8, 20 mg/kg bw.
The test item-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 (carcinogenicity) study animals. The concentration was adjusted weekly using the food consumption valued from 2 weeks earlier.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 days) 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:

2 year

Frequency of treatment:

Daily in feed

Post exposure period:

None

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

4 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

8 mg/kg bw/day (nominal)

Remarks:

in diet

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

in diet

No. of animals per sex per dose:

Chronic toxicity study (satellite study): in total 100 (50 male and 50 female): 10 animals/sex for groups 1, 2, 3 and 20 animals/sex for group 4 (high dose)

Control animals:

yes, plain diet

Details on study design:

Post-exposure period: none

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- daily individual observations for any signs of behavioural changes, reaction to treatment or illness.

DETAILED CLINICAL OBSERVATIONS: Yes
- Weekly individual observations in standard arena, including 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, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.


BODY WEIGHT: Yes
yes, on same day once per week; from week 14 intervals of two weeks.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
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) is 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).

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


OPHTHALMOSCOPIC EXAMINATION: Yes
Examinations will be performed on all main (carcinogenicity) study animals prior to dosing, in test week 52 and at the end of the main study (test week 104).

HAEMATOLOGY: Yes
After TW 13, 26, 52: All animals satellite group 1,2 and 3, and group 4 first 10 animals.
Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, prothrombin time, thromboplastin time; reticulocytes, MCV, MHV, MCHC,

CLINICAL CHEMISTRY: Yes
After TW4: first 10 animals Satellite groups 1 & 4.
After TW 13, 26, 52: all animals of satellite groups 1,2 and 3, and first 10 animals of group 4
Sodium, potassium, glucose, total cholesterol, urea, total bilirubin, creatinine, total protein and albumin, alanine aminotransferase (ALAT), aspartate amino¬transferase (ASAT), alkaline phosphatase (aP), gamma glutamyl transpeptidase (γGT).
Calcium, chloride, globulin, abumin/globulin ration, LDH.

URINALYSIS: Yes
After TW 13, 26, 52: all animals from satelite groups 1,2 and 3, and first 10 animals of group 4.
Volume, specific gravity, pH, protein, glucose, blood
Bilirubin, urobilinogen, ketones, nitrite, epithelial cells, leucocytes, erythrocytes, organisms, crystalluria, further constituents (i.e. sperm, casts).

NEUROBEHAVIOURAL EXAMINATION: Yes
In test week 52, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on GAD5), as well as the assessment of grip strength (MEYER6) and motor activity assessment were conducted in 10 satellite animals/sex/group (in total 80 animals):
- observational screen: Righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.
- Functional tests: grip strength, locomotor activity.
The auditory acuity is checked with a simple noise test on all main (carcinogenicity) study animals prior to dosing, in test week 52 and at the end of the main study (test week 104).

Sacrifice and pathology:

Organ Weights: Liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, spleen, brain, heart

Gross and histopathology: All listed tissues of all animals of groups 1 and 4 and of deceased or prematurely sacrificed animals will be microscopically examined.
Brain, spinal cord, pituitary, thyroid, parathyroid *, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, lymph node, peripheral nerve, bone marrow, skin, eyes
* The parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged.
Other: coecum, epididymis, eyes, tissue masses

Statistics:

Multiple t-test: DUNNETT, C. W. New tables for multiple comparisons with a control Biometrics, 482-491 (September 1964):
Exact test of R. A. FISHER
Body weight / food consumption / relative and absolute organ weights (p ≤ 0.01):
histopathology (p ≤ 0.01)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Mortality rates [%] Survival rates [%]
at study termination# at study termination#
Males Females Males Females
Group 1
Control 56 42 44 58
Group 2
4 mg/kg 48 68 52 32
Group 3
8 mg/kg 50 38 50 62
Group 4
20/15/12
mg/kg [80]#1 [76]#2 [20]#1 [24]#2

** statistically significant different from the control at p <= 0.01 (Fisher test)
#1 the in-life phase of the high dosed males was terminated on test day 451 (test week 65) after 64 test weeks
#2 the in-life phase of the high dosed females was terminated on test day 597 (test week 86) after 85 test weeks

The male and female animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpro-pane-1,3-diamine/kg b.w./day revealed in-creased mortality and corresponding decreased survival rates from approximately test week 46 (males, statistically significant at p ≤ 0.01 from test week 50 onwards) and from approximately test week 55 (females, statistically significant at p ≤ 0.01 from test week 62 onwards) onwards. The severely increased mortality rates noted for the high dosed animals resulted in the discontinuation of dosing from test weeks 62 (males) and 82 (females) onwards and, subsequently, in the study termination of the high dosed males in test week 65 and of the high dosed females in test week 86.

None of the rats treated with 4, 8 or 20/15/ 12 mg N-(3-aminopropyl)-N-dodecylpro-pane-1,3-diamine/kg b.w./day revealed any test item-related clinical signs of systemic toxicity during the course of the study.



BODY WEIGHT AND WEIGHT GAIN
The body weight of males and females treated with 8 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day was below the body weight of the control group from approximately test week 53 onwards in males (by up to 14%, statistically significant at p ≤ 0.01 in test weeks 59 to 91, 95 and 101) and from test week 75 onwards in females (by up to 12%, statistically not significant at p ≤ 0.01).
Treatment with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day led to a reduced body weight of males and females from approximately test week 19 onwards in males (by up to 32%, statistically significant at p ≤ 0.01 in test weeks 19 to 63) and from test week 31 onwards in females (by up to 24%, statistically significant at p ≤ 0.01 in test weeks 31, 33, 37 to 85).
The total body weight gain was accordingly reduced in the male and female animals of the intermediate and high dose group treated with 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecyl-propane-1,3-diamine/kg b.w./day when com-pared to the respective start values and to the control group.



FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related influence was noted on the food consumption of the animals treated with 4, 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day.
The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.

Test item via the diet / mean values [mg/kg b.w. per day]
Group 2 Group 3 Group 4
4 mg/kg 8 mg/kg 20/15/12 mg/kg
males females males females males females
4 mg/kg 8 mg/kg 20 mg/kg
(TW 1-104) (TW 1-104) (TW 1-50)

Max 4.4 5.0 10.1 10.0 22.5 22.2
Min 3.2 3.3 6.1 6.4 15.9 16.9
Mean 3.97 4.02 8.01 8.08 19.59 19.85
SD 0.21 0.34 0.59 0.66 1.34 1.39
15 mg/kg
(TW 51-55) (TW 51-58)
Max - - - - 17.9 16.4
Min - - - - 14.7 14.4
Mean - - - - 15.99 15.56
SD - - - - 1.67 0.89
12 mg/kg
(TW 56-61) (TW 59-81)
Max - - - - 14.6 13.3
Min - - - - 11.5 11.1
Mean - - - - 13.02 12.40
SD - - - - 1.56 0.73



FOOD EFFICIENCY: not examined


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.


OPHTHALMOSCOPIC EXAMINATION
No test item-related influence was noted.


HAEMATOLOGY
The animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day revealed a decreased haemoglobin content, haematocrit value, mean corpuscular volume (MCV) and mean corpuscular haemo-globin (MCH), increased numbers of leucocytes, reticulocytes and platelets, and several changes in the absolute and relative differential blood counts.
The changes were noted for both genders in test weeks 26 and 52, and in test weeks 78 and 86 for the remaining high dosed females.


CLINICAL CHEMISTRY
The following test item-related changes were noted in the male animals treated with 8 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/ kg b.w./day and in the male and female animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day: decreased plasma levels of glucose, total protein (and a corresponding decrease of the albumin and globulin levels), and an increased ASAT activity.
In addition, further changes were noted for the high dosed males in form of increased plasma levels of creatinine and urea, and an increased LDH activity.
The changes were noted in test weeks 13, 26, 52, 65 (group 4 males only); 78, 86 (group 4 females only) or 104 (group 3 males only).


URINALYSIS
Treatment with 20/15/12 mg N-(3-aminopropyl) -N-dodecylpropane-1,3-diamine/kg b.w./day led to a slightly decreased pH value of the urine compared to the control group in male and female animals during the course of the study.
In addition, the male animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpro-pane-1,3-diamine/kg b.w./day revealed a slightly increased amount of haemoglobin (25 to 250 ery/µL) in the urine of all 10 males examined in test week 52 compared to the control group (where only 5 of 10 males were affected).


NEUROBEHAVIOUR: not examined


ORGAN WEIGHTS
Treatment with either 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day led to an increase in the relative heart and kidney weights of males and females compared to the control animals caused by the reduced body weight of the animals. Additionally, reduced absolute heart and kidney weights were noted for the high dose group.


GROSS PATHOLOGY
A few animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day revealed test-item related changes in form of various changes of the heart (such as dilated, discoloured, enlarged, gelatinous and/or pale) and the mesenteric lymph nodes (discoloured, thickened and/or reddened) which correlated with test-item related microscopic findings in these organs.


HISTOPATHOLOGY: NON-NEOPLASTIC
Non-neoplastic lesions were noted in the heart, skeletal muscle, kidney, lymph node (mesenteric) of male and female rats which are related to the testitem:
Male and female rats treated with 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecyl-propane-1,3-diamine/kg b.w./day revealed a dose dependent mild to moderate lympho-histiocytic myocarditis with degeneration of heart muscle cells, increase of granulation tissue and fibrosis between the muscle cells. Similar changes were noted for the skeletal muscle.
In particular, for the animals treated with 20/15/12 mg N-(3-aminopropyl)-N-dodecylpro-pane-1,3-diamine/kg b.w./day a degeneration of muscle cells and granulation tissue with fibrosis were noted in the skeletal muscle of the leg and larynx. Several male and female rats treated with 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecyl-propane-1,3-diamine/kg b.w./day revealed a mild to moderate chronic nephropathy. The cardiac and renal changes correlated with the macroscopic results.
The mesenteric lymph nodes of the animals treated with 4, 8 or 20/15/12 mg N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine/kg b.w./day showed a mild to marked increase in the number of macrophages with an eosinophilic homogenous/vacuolized cytoplasm caused by an accumulation of the test item in the macrophages. This was considered to reflect the oral route of exposure.



HISTOPATHOLOGY: NEOPLASTIC
The histomorphological examination of rat organs from a long-term study by dietary administration of the test item N-(3-amino-propyl)-N-dodecylpropane-1,3-diamine did not reveal any changes in the incidence and severity of tumors which are considered to be related to the administration of the test item.

HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS
BONE MARROW EVALUATION:
The myeloid : erythroid ratio of the high dosed male and female animals was not influenced.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: toxicity

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Conclusions:

Under the study conditions, the test substance, given via the diet daily for 104 weeks, had no carcinogenic potential in rats.

Executive summary:

A study was conducted to determine the carcinogenic potential of the test substance according to OECD Guideline 453, in compliance with GLP. Male and female Crj: CD(SD) rats were treated with 4 or 8 mg/kg bw/day by dietary administration for 104 weeks or with 20/15/12 mg/kg bw/day by dietary administration for 61 weeks (high dosed male animals) or 81 weeks (high dosed female animals). A dose-related systemic toxicity was noted. A MTD (maximum tolerated dose) as defined by the ICH guideline S1C(R2): 'Dose Selection for Carcinogenicity Studies of Pharmaceuticals' in form of e.g. no more than 10% decrease in body weight gain relative to controls, target organ toxicity and/or significant alterations in clinical pathological parameters was met at the 8 mg/kg bw/day dose level. The body weight of the males and females treated with 8 mg/kg bw/day was below the body weight of the control group from approximately test week 53 onwards in males (by up to 14%, statistically significant at p ≤ 0.01 in test weeks 59 to 91, 95 and 101) and from Week 75 onwards in females (by up to 12%, statistically not significant at p ≤ 0.01). In addition, changes were noted for biochemical and haematological parameters and non-neoplastic changes were noted in the heart, kidneys, skeletal muscle of the leg and larynx and mesenteric lymph nodes. Hence, the 8 mg/kg bw/day dose was considered to be the maximum tolerated dose suitable for histopathological evaluation for neoplastic changes. All neoplastic lesions recorded in this study were commonly encountered in rats of this strain and age. Type, incidence, and severity of the lesions recorded were not increased in the test-item treated animals as compared to the control animals. Under the study conditions, the test substance, given via the diet daily for 104 weeks, had no carcinogenic potential in rats (Hansen, 2011).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on a dietary carcinogenicity study conducted in rats, the substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.

Additional information

A study was conducted to determine the carcinogenic potential of the test substance according to OECD Guideline 453, in compliance with GLP. Male and female Crj: CD(SD) rats were treated with 4 or 8 mg/kg bw/day by dietary administration for 104 weeks or with 20/15/12 mg/kg bw/day by dietary administration for 61 weeks (high dosed male animals) or 81 weeks (high dosed female animals). A dose-related systemic toxicity was noted. A MTD (maximum tolerated dose) as defined by the ICH guideline S1C(R2): 'Dose Selection for Carcinogenicity Studies of Pharmaceuticals' in form of e.g. no more than 10% decrease in body weight gain relative to controls, target organ toxicity and/or significant alterations in clinical pathological parameters was met at the 8 mg/kg bw/day dose level. The body weight of the males and females treated with 8 mg/kg bw/day was below the body weight of the control group from approximately test week 53 onwards in males (by up to 14%, statistically significant at p ≤ 0.01 in test weeks 59 to 91, 95 and 101) and from Week 75 onwards in females (by up to 12%, statistically not significant at p ≤ 0.01). In addition, changes were noted for biochemical and haematological parameters and non-neoplastic changes were noted in the heart, kidneys, skeletal muscle of the leg and larynx and mesenteric lymph nodes. Hence, the 8 mg/kg bw/day dose was considered to be the maximum tolerated dose suitable for histopathological evaluation for neoplastic changes. All neoplastic lesions recorded in this study were commonly encountered in rats of this strain and age. Type, incidence, and severity of the lesions recorded were not increased in the test-item treated animals as compared to the control animals. Under the study conditions, the test substance, given via the diet daily for 104 weeks, had no carcinogenic potential in rats (Hansen, 2011).