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-

Endpoint summary

Description of key information

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

As no adverse effects were found, the highest dose tested of 80 mg/kg bw/day was determined as NOAEL for systemic toxicity. 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

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

Specific details on test material used for the study:

pH: 10.2-10.7 at concentration of 1%

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

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.

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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

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

Critical effects observed:

no

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality data (GLP an dguideline studies) with results that are consistent witrh other data available for the group of polyamines.

System:

gastrointestinal tract

Organ:

mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Oleyl tripropylenetetramine is a paste with mp of 31°C and has a vapour pressure of less than 4.7 x 10-5 Pa at 20°C (Based on read-across on coco-triamine, a similar product with shorter chain lengths). Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Oleyl tripropylenetetramine is a paste with mp of 56 °C and has a vapour pressure of less than 4.7 x 10-5 Pa at 20°C (Based on read-across on coco-triamine, a similar product with shorter chain lengths). Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur. In view of corrosive properties, exposures via inhalation when occurs would be characterised by local irritation.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Oleyl tripropylenetetramine is corrosive to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Oleyl tripropylenetetramine is corrosive to the skin.

Mode of Action Analysis / Human Relevance Framework

The most significant treatment-related changes in all studies performed on polyamines are effects on the small intestine and mesenteric lymph nodes. A relatively strong inflammatory reaction is also observed at high dose levels. These effects in the gastro-intestinal tract have consistently been observed with these polyamines.

A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. The observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and generally considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance, and for substances belonging to the same group of chemicals, and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect.

Phospholipidosis is a plausible mechanism. In physiological circumstances, the polyamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil. The complex of cationic surfactant and phospholipids are difficult to digest by the macrophages, and they accumulate with the lysosomes. Recent (unpublished) studies have shown that these cationic surfactants are lysosomotropic, and scored positive for phospholipidosis inin vitrostudies with HepG2 cells.

Additional information

Oral:

A repeated dose 28-day oral toxicity study with Oleyl 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 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.

Oleyl tripropylenetetramine formulated in propylene glycol, was administered daily for 28 days by oral

gavage to groups of 5 male and 5 female SPF-bred Wistar rats.

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. Consequently, the report concludes to a NOAEL 80 mg/kg bw/day.

In the attached illustration, a graph is presented in which the (semi-quantified) effects involving foamy macrophages & Sinus histiocytosis from this study on Oleyl tripropylenetetramine and that from the OECD 422 study on Tallow (C16-18, C18-unsat.) tripropylenetetramine. As can be observed, they are actually very much in-line with each other. In the Tallow tripropylenetetramine, the 30 mg/kg was considered a LOAEL based on foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci found in the mesenteric lymph nodes, a lower BW, and higher relative neutrophil count with a corresponding reduction in relative lymphocyte count in females. In comparison: In only 4 out of 24 available studies now there was a little foamy macrophages also observed in control, with a highest level corresponding to 2.5 in the graph below.

Therefore, based on observed foamy macrophages, although not considered adverse, the NOAEL for this 28-day study on Oleyl tripropylenetetramine is set on 20 mg/kg bw/day, which is more in-line with the other available data.

Cross-reading and category approach:

Within the category of the Polyamine there are further studies available that clearly demonstrate the same toxicological profile for the various substances over this category, and also give some rules of thump to be considered when cross-reading between group members.

(See the document for the category justification for the polyamines that is attached to IUCLID Ch. 13.).

 

A repeated dose 90-day oral toxicity study with C16-18, C18-unsaturated-alkyl dipropylene triamine (Tallow dipropylenetriamine) was performed according to OECD 408 guidelines and GLP principles.Male and female rats (10/sex/exposure) were exposed to 0, 2.5, 10, 40 or 160 mg/ kg bw/ day by daily gavage. Based on the high mortality rate and signs of deteriorated health condition of animals at 160 mg/kg bw/ day, this high dose group was terminated after 11 weeks of dosing. At 40 mg/kg bw/day, 3/10 males were killed in extremis in the last week of treatment. Rats treated with 40 and 160 mg/kg bw/day showed clinical signs including a hunched posture, piloerection, pallor, and swelling of the abdomen and/or legs, and an abnormal posture or gait, as well as reduced motor activity at 160 mg/kg bw/day. A lower body weight gain and food intake was recorded for males at 40 and 160 mg/kg (body weights were approximately 11 and 23% lower than controls at the end of treatment, respectively). At 10 mg/kg bw/day one male was found dead in Week 12. This animal displayed no clinical signs prior to death and had normal body weight gain, however it is not clear if this death was substance-related. Below 40 mg/kg bw/day, no clinical signs or changes in functional behaviour were noted.

The NOAEL was determined at 2.5 mg/kg bw/day, based on effects observed at 10 mg/kg bw/day. At this level changes in blood were confined to a higher inorganic phosphate level, and marginally higher neutrophil counts. Also at 10 mg/kg bw/day, adverse histopathological findings were observed in the jejunum/ileum (minimally to slight foamy macrophages in lamina propria), mesenteric lymph nodes (general minimal to moderate foamy macrophages, accompanied by incidental cases of granulomas with central necrosis, lymphoid hyperplasia, sinus ectasia), liver (granulomatous lesion with central necrosis in 2 males) and bone marrow (minimal myeloid hyperplasia in 7/40 animals).

 

Further supportive information comes from read-across to other repeated dose studies in rat on Polyamines:

Sub-group	Alkyl chain	Study	Results (in mg/kg bw/d)
Diamines	C12/14	28-day study (OECD 407)	NOAEL: 0.4 mg, based on moderate accumulations of foam cells in the jejunum and ileum, foam cell infiltration in mesenteric lymph node.
		90-day (OECD 408)	NOAEL: 0.4 mg, based on enteropathy with foam cell accumulation in the small intestine and mesenteric lymph node with accompanying inflammatory changes seen in the mid and high dose groups. Not reversible during 90-day recovery period.
	Oleyl	14-day range finder	NOAEL: 2 mg, enteropathy with foam cell accumulation in the small intestine and mesenteric lymph node with accompanying inflammatory changes seen in the mid and high dose groups.
		28-day (OECD 408)	NOAEL: 1.25 mg, based on enteropathy with foam cell accumulation in the small intestine and mesenteric lymph node with accompanying inflammatory changes seen in the mid and high dose groups. Not reversible during 14 day recovery period.
Triamines	Coco	OECD 422	LOAEL: 10 mg, 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.
	Tallow	90-day (OECD 408)	NOAEL 2.5 mg/kg bw/day based on foamy macrophage infiltration in the ileum and jejunum.
Y-triamines	C12	90-day (OECD 408)	NOAEL = 7-8 mg (m-f, dietary level at 100 ppm), based on enlargement of mesenteric lymph nodes with foamy macrophages, decreased BW gain, increased ASAT and ALAT levels, and tubular nephropathy at higher dose level.
		Chronic/carc (OECD 453)	NOAEL = 4 mg (dietary level ca. 100 ppm) based on increased ASAT, increase of lymphohistiocytic inflammatory reactions in the kidney, skeletal muscle and heart, and foamy macrophages in the mesenteric lymph nodes and alveoli of the lungs at next dose level.
Tetramines	Tallow	OECD 422	LOAEL = 30 mg, 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.
	Oleyl	28-day (OECD 407)	NOAEL 80 mg for systemic toxicity. However, based on observed foamy macrophages the NOAEL is set on 20 mg/kg bw/day.

 

 

The most significant treatment-related changes in all studies performed on polyamines are effects on the small intestine and mesenteric lymph nodes. A relatively strong inflammatory reaction is also observed at high dose levels. These effects in the gastro-intestinal tract have consistently been observed with these polyamines, and are considered local effects related to the corrosive nature of the substances.

A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. It is indicative that the observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance, and for substances belonging to the same group of chemicals, and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect. In that respect, considering that macrophages in the mesenteric lymph nodes are a local, porte d’entrée related effect due to the route of application, and not a systemic effect per se, the level of 2.5 mg/kg bw/day for Tallow dipropylenetriamine could also be argued to represent a NOAEL for local effects, and that the NOAEL for systemic effects is higher.

 

Dermal:

For dermal exposure no good overall NOAEL can be established as effects are rather characterized by local corrosive effects that are related to duration, quantity and concentration, than by systemic toxicity due to dermal uptake. The mode of action of for polyamines follows from its structure, consisting of an apolar fatty acid chain and a polar end of a primary amine linked to a secondary amine. The structure can disrupt the cytoplasmic membrane, leading to lyses of the cell content and consequently the death of the cell. The polyamines are completely protonated under environmental conditions which causes them to strongly adsorb to organic matter. This all leads to a low dermal absorption as is shown by a dermal absorption study performed on a structurally related branched triamine (Dodecyl dipropylene triamine) for 24 hours, resulted in a dermal penetration of less than 0.01% whereas 0.92% of the applied dose did pass the stratum corneum but remained further fixed in the skin. For the linear polyamines a similar and even lower (in case of higher alkyl chain lengths) dermal penetration can be expected.

 

An indication of the threshold for irritation can be obtained from a sensitisation study on one of the higher corrosive substance within PPA group: 0.5% in distilled water was found to be non-irritant (0.5 mL, 6 hrs, occlusive) on rabbit skin.

 

Inhalation:

Physical-chemical properties of Oleyl tripropylenetetraamine indicate a low likelihood for exposure via inhalation, with a melting point of 56 °C, a boiling point > 300 °C and low vapour pressure (4.7 x 10-5 Pa at 20°C for the coco dipropylene triamine, with the shortest average alkyl chain length representing the highest vapour pressure for the group of polyamines).

Justification for classification or non-classification

Classification for STOT-RE Cat. 2 is required in case of significant toxic effects at levels ≤ 100 mg/kgbw/d in case of standard 90-day study. In case of 28-day studies this can be multiplied by 3.

STOT-RE Cat.1 is required in case of significant toxicity at levels at levels ≤ 10 mg/kgbw/d in 90-day studies or ≤ 30 mg/kgbw/d in case of 28-day studies.

 

For this endpoint 'repeated dose toxicity by oral route' results from a 28-day repeated dose study is available, resulting to a NOAEL of 20 mg/kg bw based on foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci found in the mesenteric lymph nodes observed at 80 mg/kg dose level. However, since these effects at 80 mg/kw were in the absence of clear signs of degeneration, necrosis or accompanying inflammatory changes, the study report concluded that the effects observed were not adverse and only represent adaptive changes.

 

The OECD 422 study on Tallow tripropylenetetraamine indicted severe toxicity at 300 mg/kg causing the termination of the whole dose group making it eligible for Cat.2 classification. At 30 mg/kg/day, no severe toxicity was observed, and consequently, the criteria for GHS STOT-RE Cat.1 classification are not met. At 100 mg/kg/day also 2 animals had to be killed in extremis, which in one animal was related with gastro-intestinal corrosion.

 

In conclusion: Available data results to classification for STOT-RE: Cat.2 [Warning]; H373: May cause damage to organs through prolonged or repeated exposure (based on mortality). Affected organs: Gastro intestinal system- Mesenteric lymph nodes.