Reaction mass of N-[3-(dimethylamino)propyl]docosanamide and N-[3-(dimethylamino)propyl]stearamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-09 to 2012-05-30

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]stearamide

EC Number:

231-609-1

EC Name:

N-[3-(dimethylamino)propyl]stearamide

Cas Number:

7651-02-7

Molecular formula:

C23H48N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]octadecanamide

Test material form:

solid: pellets

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: app. 6 weeks
- Housing: group housing of 3 animals per sex in Macrolon cages with sterilised sawdust as bedding material and paper as cage-enrichment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
formulations were placed on a magnetic stirrer during dosing;
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.

Dose volume: 5 mL/kg bw, actual dose volumes were calculated weekly according to the latest body weight

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at testing laboratory and on information from the sponsor.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No chemical analysis of formulations were performed in the 14-day range finding study. Instead, analysis on formulations will be performed as part of the subsequent repeated dose toxicity study.

Duration of treatment / exposure:

14days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Initially, one dose group at 500 mg/kg bw/d was initiated (next to a concurrent control group). Based on the results obtained at 500 mg/kg bw/d, two additional doses at 50 and 200 mg/kg bw/d were added one week later.
- Rationale for animal assignment (if not random): animals were allocated at random, with all animals within ± 20% of the sex mean
- Rationale for selecting satellite groups: no satellite groups were used in the dose range finding study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily; animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards, detailed clinical observations were made in all animals at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 4, 7, 10 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- over days 1-4, 4-7, 7-10 and 10-14

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

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 specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals at 500 mg/kg bw/d were sacrificed for humane reasons between Days 6 and 8.
No mortality occurred at 50 and 200 mg/kg bw/d.

500 mg/kg bw/d:
- lethargy, hunched posture, labored respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards

200 mg/kg bw/d:
- all animals showed piloerection on two days of week 2 only

50 mg/kg bw/d:
- no clinical signs were noted

Salivation seen after dosing among all animals at 200 and 500 mg/kg bw/d on a few days of treatment was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).

BODY WEIGHT AND WEIGHT GAIN
500 mg/kg bw/d
- two females showed weight loss between days 1 and 4 (2 or 5% compared to day 1), followed by a slight weight gain between days 4 and 7. One female and all males at 500 mg/kg bw/d showed a reduced weight gain throughout the treatment period.

At 50 and 200 mg/kg, body weights and body weight gain remained in the same range as controls over the study period (the animals used for dosing at 50 and 200 mg/kg bw/d were from a different batch of delivery, and hence the starting body weight on day 1 was different to that for control animals and animals at 500 mg/kg bw/d).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
500 mg/kg bw/d:
- food consumption before or after correction for body weight was reduced in both genders over days 1-4 and 4-7

200 mg/kg bw/d:
- food consumption before or after correction for body weight was similar to controls over the study period

50 mg/kg bw/d:
- food consumption before or after correction for body weight was similar to controls over the study period

HAEMATOLOGY
The following changes in haematology parameters distinguished treated from control animals and from normal range levels encountered for rats of this age and strain:
- Slightly lower red blood cell counts in males at 50 and 200 mg/kg bw/d (no clear dose related trend).
- Higher reticulocyte counts in males at 50 and 200 mg/kg bw/d (no clear dose related trend).

CLINICAL CHEMISTRY
The following changes in clinical biochemistry parameters distinguished treated animals from control animals and from normal range levels encountered for rats of this age and strain:
-higher alanine aminotransferase activity (ALAT) in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d
-Higher alkaline phosphatase activity (ALP) in one female at 200 mg/kg bw/d
-Higher potassium level in males at 50 and 200 mg/kg bw/d


ORGAN WEIGHTS
Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group.

The apparent lower liver, spleen and heart weight of males at 200 mg/kg bw/d was ascribed to a slightly lower terminal body weight as the ratio to body weight for these organs was similar to control levels.

GROSS PATHOLOGY
500 mg/kg bw/d:
- reduced size of seminal vesicles, prostate, epididymides, spleen and/or thymus, gelatinous contents in the gastro-intestinal tract, small intestines and/or caecum, gastro-intestinal tract distended with gas, and emaciated appearance

200 mg/kg bw/d:
- no abnormalities

50 mg/kg bw/d:
- no abnormalities

HISTOPATHOLOGY: NON-NEOPLASTIC
500 mg/kg bw/d:
-Thymus: Lymphoid atrophy in 2/2 females (moderate).
-Stomach: Hyperplasia of the forestomach in 3/3 males and 3/3 females at slight or moderate degree, inflammation of the forestomach in 3/3 males and 3/3 females up to moderate degree, ulceration of the forestomach in 1/3 males and 1/3 females at slight degree.
-Duodenum: Hyperplasia of the villi in 2/3 males and 2/3 females up to slight degree.
-Jejunum: Hyperplasia of the villi in 1/3 females at minimal degree.
-Mesenterial lymph node: Foamy macrophages and sinusoidal dilation in 3/3 males and 3/3 females at slight or moderate degree, and congestion/erythrophagocytosis in 1/3 males and 2/3 females at minimal degree.
-Testes: Absence of spermiation (massive degree) and degeneration of spermatids in 3/3 males up to slight degree.
-Epididymides: Oligospermia in 2/2 males at massive degree and seminiferous cell debris in 2/2 males at slight degree.
-Prostate: Reduced contents in 2/2 males at slight degree.
-Seminal vesicles: Reduced contents in 2/2 males at moderate degree.

No treatment-related microscopic abnormalities were noted at 50 and 200 mg/kg bw/d.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

All animals at 500 mg/kg bw/d were sacrificed for humane reasons between Days 6 and 8, and showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from Day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included:

-     Lymphoid atrophy of the thymus in 2/2 females examined, correlating to a reduced size of the thymus at necropsy.

-     Hyperplasia and inflammation (3/3 males and 3/3 females) and ulceration (1/3 males and 1/3 females) of the forestomach.

-     Hyperplasia of the villi in the duodenum (2/3 males and 2/3 females) and jejunum (1/3 females).

-     Foamy macrophages and sinusoidal dilation (3/3 males and 3/3 females) and congestion/ erythrophagocytosis (1/3 males and 2/3 females) in the mesenterial lymph node.

-     Absence of spermiation and degeneration of spermatids in the testes in 3/3 males, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles in 2/2 males examined, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

 

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of Week 2 only, whilstno clinical signs were noted at 50 mg/kgbw/d.Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

 

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

 

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d.

Applicant's summary and conclusion

Conclusions:

In a 14 d dose range finding subacute toxicity study, Stearic acid 3-(dimethylaminopropyl)amide was administered to 3 Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.
All animals of the highest dose group were sacrificed for humane reasons based on clinical signs. The main cause for moribundity was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach.
In the 50 and 200 mg/kg bw/d dose groups, no mortality occurred, and no or only minor clinical signs were observed. No treatment related effects were observed at necropsy or histopathology.

Executive summary:

In a 14 d dose range finding study according to OECD guideline 407, adopted 03 October 2008, and EU method B.7, May 2008, Stearic acid 3-(dimethylaminopropyl)amide was administered to 3Crl:WI(Han) rats/sex/dose orally via gavage at dose levels of 0, 50, 200 and 500 mg/kg bw/day.

All animals in the 500 mg/kg bw/d dose group were sacrificed for humane reasons between days 6 and 8. They showed lethargy, hunched posture, laboured respiration, abdominal swelling, piloerection, chromodacryorrhoea, a lean appearance and/or ptosis from day 4 of treatment onwards. All animals showed weight loss or reduced body weight gain and reduced food consumption during the treatment period. Necropsy findings at 500 mg/kg bw/d primarily consisted of gelatinous contents in the gastro-intestinal tract or parts thereof, and emaciation. The main cause for moribundity at this dose level was forestomach ulceration and/or hyperplasia of the squamous epithelium of the forestomach. Other histopathological changes noted at this dose level included: lymphoid atrophy of the thymus, correlating to a reduced size of the thymus at necropsy; hyperplasia and inflammation of the forestomach; hyperplasia of the villi in the duodenum and jejunum; foamy macrophages and sinusoidal dilation and congestion/ erythrophagocytosis in the mesenterial lymph node; absence of spermiation and degeneration of spermatids in the testes, oligospermia and seminiferous cell debris in the epididymides, and reduced contents in the prostate and seminal vesicles, which corresponded to a reduced size of seminal vesicles, prostate and epididymides at necropsy.

 

At 50 and 200 mg/kg bw/d, no mortality occurred. All animals at 200 mg/kg bw/d showed piloerection on two days of week 2 only, whilstno clinical signs were noted at 50 mg/kgbw/d.Body weights, body weight gain and food intake remained in the same range as controls over the study period at these dose levels.

 

At 50 and 200 mg/kg bw/d, haematological changes consisted of slightly lower red blood cell and higher reticulocyte counts in males. No clear dose related trend was noted for these changes, which were generally slight in nature. Clinical biochemistry changes consisted of higher alanine aminotransferase activity in two males at 50 mg/kg bw/d, and two males and one female at 200 mg/kg bw/d, higher alkaline phosphatase activity in one female at 200 mg/kg bw/d, and higher potassium level in males at 50 and 200 mg/kg bw/d.

 

At 50 and 200 mg/kg bw/d, no abnormalities were noted at necropsy. Spleen and thymus weights of females at 200 mg/kg bw/d appeared slightly increased compared to the control group. No treatment-related histopathological changes were noted at 50 and 200 mg/kg bw/d.

 

Based on these results, dose leves were selected for the reproduction/developmental toxicity screening test according to OECD guideline.