Amines, N-(3- aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-

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:

03 October 2016 - 02 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (28-day Oral Toxicity Study in Rodents)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Location 's-Hertogenbosch d.d. 3 November 2017 and location Schaijk d.d. 13 February 2017

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-(3- aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-

EC Number:

701-369-7

Molecular formula:

UVCB - No molecular formula;

IUPAC Name:

Amines, N-(3- aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-

Constituent 2

Reference substance name:

(Z)-N-(3-aminopropyl)-N'-[3-(9-octadecenylamino)propyl]propane-1,3-diamine

EC Number:

266-613-2

EC Name:

(Z)-N-(3-aminopropyl)-N'-[3-(9-octadecenylamino)propyl]propane-1,3-diamine

Cas Number:

67228-83-5

Molecular formula:

C27H58N4

IUPAC Name:

N-[3-[3-[[(E)-octadec-9-enyl]amino]propylamino]propyl]propane-1,3-diamine

Test material form:

other: paste

Details on test material:

- Name of test material (as cited in study report): Oleyl (vegetable oil) tripropylenetetramine
- Appearance: White paste
- Storage condition of test material: At room temperature container flushed with nitrogen
- pH: 10.2-10.7 at concentration of 1%

Specific details on test material used for the study:

pH: 10.2-10.7 at concentration of 1%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Males 141 - 168 gram and females 120 - 138 gram
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Set to maintain:
- Temperature (°C): 18 – 24 (range of actual daily mean: 22.0 - 23.3°C)
- Humidity (%): 40 - 70 (range of actual daily mean: 44-63%)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 03 October 2016 to 02 November 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

This study should provide a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

propylene glycol

Remarks:

s.g. 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. The formulations were heated to a maximum of 61.3°C for a maximum of 31 minutes to obtain visual homogeneity. Formulations were released for dosing when they had obtained a temperature of 40°C or lower. Adjustment was made for specific gravity of the vehicle. No correction was made for purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at Charles River Den Bosch and on information provided by the Sponsor.

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The determination of Oleyl (vegetable oil) tripropylenetetramine in formulation samples was performed using LC-MS/MS.
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 – 110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference between the stored and freshly taken samples was ≤ 10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected at 0, 5, 20 and 80 mg/kg bw/day by the Sponsor based on the available data on the very comparable substance Tallow tripropylenetetramine (C16-18, C18- unsaturated-alkyl tripropylenetetramine, CAS 1219458-11-3), for which a combined repeated dose/developmental toxicity screening study (OECD 422) was available, applying dose levels 0, 30, 100, 300 mg/kgbw/day (Notox, study 91255, 2010). This study resulted in a LOAEL of 30 mg/kg bw/day derived based on foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci found in the mesenteric lymph nodes observed at this dose level, and premature termination of 300 mg/kg bw dose-group after 10 days due to excessive mortality. The 100 mg/kg bw resulted in mortality of one male and female and lower body weights lower body weights compared to control.

- Randomization:
By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, immediately (0-15 minutes) after dosing, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment phase, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- 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 (for a maximum of 24 hrs)
- How many animals: All
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes (for a maximum of 24 hrs)
- How many animals: All
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 of treatement
- Dose groups that were examined: All
- Battery of functions tested: sensory reactivity (auditory, visual and proprioceptive stimuli), grip strength and motor activity according to test guidelines

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were fasated overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: All
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY: Yes
According to test guidelines

The following slides were examined by a pathologist:
- All tissues collected at the scheduled sacrifice from all Group 1 and 4 animals
- The jejunum, ileum and mesenteric lymph nodes of all males and females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4
- All gross lesions

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Occasionally rales were noted for two male and two female animals treated at 80 mg/kg bw/day. This was not considered toxicologically relevant, considering the frequency of occurrence and minor severity of the effect.
Salivation was noted on most occasions after dosing at all dose levels with a dose related incidence. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A trend towards a lower body weight gain was shown by females treated at 80 mg/kg bw/day during the second half of the study. This was considered not toxicologically significant since the weights were within the expected range and taking into account the nature of the effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A trend towards a lower food consumption before and after correction for body weight for females treated at 80 mg/kg bw/day was shown. This was considered not toxicologically significant, taking into account the nature of the effect and since the values were within the expected range.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower red blood cells counts, haemoglobin and haematocrit in males and higher activated partial thromboplastin time (APTT) in females at 80 mg/kg bw/day, distinguished treated from control animals but were slight in nature and in the absence of corroborative findings it was considered that these changes were not toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alanine aminotransferase activity (ALAT) was increased in both sexes at 80 mg/kg bw/day compared to control animals. In addition, lower total protein, lower albumin and slightly increased total bilirubin (not statistically significant) was observed in males at 80 mg/kg bw/day compared to controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the jejunum, ileum and mesenteric lymph nodes.
Vacuolated macrophages were observed in lamina propria in 1/5 males (slight) and 1/5 females (minimal) of the jejunum in the 80 mg/kg bw/day group. In the ileum, foamy vacuolated macrophages in lamina propria were observed in 4/5 males and 5/5 females at 20 mg/kg bw/day and in 3/5 males and 2/5 females (minimal) and in 1/5 males and 3/5 females (slight) at 80 mg/kg bw/day.
In the mesenteric lymph node an increased incidence and severity of macrophage foci (in the (para)cortex) was observed in animals of the 20 and 80 mg/kg bw /day group. Minimal macrophage foci (in the (para)cortex) were observed in 1/5, 2/5 and 1/5 animals of both sexes in the 5, 20 and 80 mg/kg bw/day treatment groups, respectively. Slight macrophage foci (in the (para)cortex) were found te be present in 1/5 males and 2/5 females at 20 mg/kg bw/day and in 4/5 animals of both sexes at 80 mg/kg bw/day. In two females of the 20 mg/kg bw/day group and in all animals of the 80 mg/kg bw/day group the macrophages involved often had foamy vacuolated cytoplasm. In addition, minimal sinus histiocytosis was observed in 1/5 males and 2/5 females, slight sinus histiocytosis in 3/5 males and 3/5 females and moderate sinus histiocytosis in 1/5 males of the 80 mg/kg bw/day group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Analytical verification of doses

In total, 20 samples were included in this study. Samples for Group 1 and Group 3 were taken in duplicate from the middle position of the container and samples for treatment Groups 2 and 4 were taken in duplicate from the top, middle and bottom position.

In the Group 1 formulation, no test item was detected. The mean accuracies of the formulations of Groups 2, 3 and 4 were 98.5%, 96.4% and 101.6%, respectively and in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).

The coefficient of variation for the formulations of Group 2 and Group 4 were 1.1% and 1.0%, respectively and were considered homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of -2.4% and -1.0%, respectively. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Applicant's summary and conclusion

Conclusions:

In a 28-day oral repeated dose toxicity study with rats according to OECD/EC guidelines and in compliance with GLP principles, the NOAEL for Oleyl (vegetable oil) tripropylenetetramine was determined to be 80 mg/kg bw/day based on the absence of adverse effects at the highest dose level in this study. However, based on observed foamy macrophages in jejunum, ileum and mesenteric lymph nodes, although not considered adverse, the NOAEL is set on 20 mg/kg bw/day.

Executive summary:

A repeated dose 28-day oral toxicity study with Oleyl (vegetable oil) tripropylenetetramine by daily gavage in the rat was performed according to OCED guideline 407 and in compliance with GLP.

 

The dose levels were selected at 0, 5, 20 and 80 mg/kg by the Sponsor based on the available data on the very comparable substance Tallow tripropylenetetramine (C16-18, C18-unsaturated-alkyl tripropylenetetramine, CAS 1219458-11-3), for which a combined repeated dose/developmental toxicity screening study (OECD 422) was available, applying dose levels 0, 30, 100, 300 mg/kgbw/day (Notox, study 91255, 2010). This study resulted to a LOAEL of 30 mg/kg bw/day was derived based on foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci found in the mesenteric lymph nodes observed at this dose level, and premature termination of 300 mg/kg bw dose-group after 10 days due to excessive mortality. The 100 mg/kg bw resulted to mortality of one male and female and lower body weights lower body weights compared to control.

 

Study outline

The test item, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

 

Evaluated parameters

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours.

No toxicologically significant changes were noted in the following parameters investigated in this study (i.e. clinical signs, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination and organ weights).

Histopathological examination showed no adverse effects as a result of treatment across all doses, and the test item was well tolerated. Despite this, treatment at dose levels of 20 and 80 mg/kg resulted in accumulation of most probably the test item or derivatives within cells of the phagocytic system of the small intestines and draining mesenteric lymph node. However, since this accumulation was found in the absence of clear signs of degeneration, necrosis or accompanying inflammatory changes, it can be concluded that the effects observed were not adverse and only represent adaptive changes.

 

Conclusion

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for Oleyl (vegetable oil) tripropylenetetramine of 80 mg/kg was established. However, based on observed foamy macrophages in jejunum, ileum and mesenteric lymph nodes, although not considered adverse, the NOAEL is set on 20 mg/kg bw/day.