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Reference
Endpoint:
basic toxicokinetics, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 December 2016 till 04 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
absorption
excretion
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
Version July 22, 2010
Deviations:
no
Remarks:
Tissue distribution and metabolism were not investigated as these were not objectives of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Non-radiolabeled Test Item: di-C16-C18 (evennumbered) alkyl tripropylenetetramine, batch 1330539

Radiolabeled test item:
Identification: [octadecyl-1-14C]Tetrameen 2HT Main component
Appearance: Light brown waxy solid
Batch 9448JRD062-1
Radiochemical purity: 93.4%
Chemical purity: Not indicated
Test substance storage: In freezer (≤ -15°C)
Stable under storage conditions: Not indicated
Specific activity: 2025 MBq/mmol, 2.91 MBq/mg
Supplier: Selcia Limited, Fyfield Business and Research Park, Fyfield Road, Ongar, Essex, CM5 0GS, UK
Total activity received: 1382.5 MBq
Date received: 09 November 2016

The guidelines indicated that radiochemical purity should be ideally greater than 95%, but despite considerable efforts it was not possible to attain a greater radiochemical purity than 93.4%.
The radiochemical purity was determined by the supplier and was also checked at Charles River Den Bosch before the start of the experiment.
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Rat strain was Crl:WI(Han) (outbred, SPF-Quality), obtained from Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France. Young adult animals of 8-12 weeks old were used. Environmental controls for the animal room were set to maintain 18 to 24°C (range of actual daily mean: 20.2-20.6°C), a relative humidity of 40 to 70% (range of actual daily mean 37-48%), approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Animals were acclimatized for at least 5 days under laboratory conditions. Animals were dosed orally at least twice during the acclimatization period with milli-Q in order to train the animals. For bile cannulated animals the acclimatization period was kept as short as possible and no oral training was performed. The day prior to dosing and following radioactive dose administration, the rats of Groups 1-2 were individually housed in stainless steel metabolism cages with a Plexiglas lid. A stream of air, which was aspired by a vacuum source, was successively passed through the metabolism cage, and through 4 glass gas absorption towers. The first two gas absorption towers contained a 1.5 M NaOH solution, to collect expired CO2 and water soluble volatiles. The next two gas absorption towers contained approximately 150 mL of Cellosolve to collect residual organic volatiles. The urine and feces collection assembly of the metabolism cage was cooled using dry ice. Following radioactive dose administration, the rats of the bile groups (Group 5) were individually housed in stainless steel metabolism cages (LxWxH = 18.5x19x20 cm) with a grid. Following radioactive dose administration, animals in Groups 3 and 4 were housed individually in Makrolon cages (type MII), equipped with a bottom grid and paper bedding. Throughout the study, except for approximately 18 hours prior to and for approximately four hours after dose administration, animals had free access to pelleted rodent diet and to tap-water.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Oral gavage using a stainless steel stomach tube. Dose volume 5 mL/kg body weight.
Formulation dose group 1, 2, 3 and 4:
An empty container with stirring device was weighed and 19.2 mg of 208027/A and 5.6017 g of 206646/B were added. Subsequently, 20.7360 g of corn oil was added and the formulation was stirred until complete dissolution. The resulting formulation was a yellow suspension.
Formulation dose group 5
An empty container with stirring device was weighed and 5.6 mg of 208027/A and 1.6002 g of 206646/B were added. Subsequently, 5.8953 g of corn oil was added and the formulation was stirred until complete dissolution. The resulting formulation was a yellow suspension.
Duration and frequency of treatment / exposure:
Groups 1, 3 and 5 were dosed once on Day 1. Groups 2 and 4: Rats were treated with unlabeled test item for 7 days prior to administration of a single radiolabeled dose.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
The dose level of 1000 mg/kg body weight was selected based on the OECD-422 Combination 28-Day Study (Test Facility Study No. 509313/509314) where no clinical signs were noted in the animals. In addition, 1000 mg/kg body weight is the limit dose according to the OECD-417 guidelines.
The radioactive dose administered to each animal was calculated from the weight of the dose administered and the mean radioactive concentration of the formulations. Animals in Groups 1 - 4 received an average dose ranging between 951 - 968 mg/kg di-C16-C18 (evennumbered) alkyl tripropylenetetramine. Animals in Group 5 received a slightly higher effective average radioactive dose of 977 mg/kg di-C16-C18 (evennumbered) alkyl tripropylenetetramine.
No. of animals per sex per dose:
4 male animals for each of groups 1, 2, 3, 4 and 5.
Positive control:
No, not relevant.
Details on study design:
Please refer to attached background material, which starts with a Table providing an outline of the study design.
Details on dosing and sampling:
Please refer to attached background material, which starts with a Table providing an outline of the study design including the duration of sampling. ADME group (Groups 1-2): The duration was 168 hours. Toxicokinetics group (Group 3-4): The duration was 24 hours. Bile-cannulated group (Group 5): The duration was 48 hours as almost 100% of the labeled test item was already excreted via feces within 48 hours in Group 1.

SAMPLING FOR 14C-ANALYSIS
Mass-balance groups (Groups 1 and 2):
Urine was collected over the following time intervals: 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168h. Feces was collected over the following time intervals: 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168h. Volatiles were collected over the following time intervals: 0-24 and 24-48h.
Bile group (Group 5):
Urine was collected over the following time intervals: 0-6, 6-24 and 24-48h. Bile was collected over the following time intervals: 0-6, 6-24 and 24-48h. Feces was collected over the following time intervals: 0-24 and 24-48h.
At termination, the interior of the metabolism cages were rinsed with methanol/water (1:1, v/v). Volatiles were collected for 48 h. Cellosolve-traps were omitted, since the daily amount collected in these traps was negligible (< 0.4% of the administered dose).
For animals in Group 3 and 4, urine, feces, bile, volatiles and cage washings were not sampled for analysis.
At the end of the collection period, the animals were euthanized and the carcass was stored at ≤-15ºC for total 14C analysis.

Toxicokinetic Blood Sampling
Approximately 0.3 mL blood samples were taken from the jugular vein and collected into
tubes containing Li-heparin as anticoagulant. Samples were taken on each of the following time points:
Group 3: 1, 2, 4, 7 and 24 hours after dosing.
Group 4: predose, 1, 2, 4, 7 and 24 hours after the last dosing.
A sub sample of approximately 0.1 mL of the blood was removed for total radioactivity analysis and the remaining blood was centrifuged to obtain the plasma.
Statistics:
Standard calculation of mean, standard deviation and coefficient of variation.
Preliminary studies:
Not applicable.
Type:
absorption
Results:
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) after a single oral dose of 1000 mg/kg bw (group 1) was 0.663% AR.
Type:
absorption
Results:
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) after a repeated oral dose of 1000 mg/kg bw (group 2) was 0.748% AR.
Type:
excretion
Results:
The mean total excretion after a single oral administration of 1000 mg/kg bw (group 1) was 108% after 7 days (0.15% in urine and 108% in faeces). Excretion in bile of bile-cannulated rats (group 5) was 0.006-0.007%.
Type:
excretion
Results:
The mean total excretion after repeated oral administration of 1000 mg/kg bw (group 2) was 101% after 7 days (0.15% in urine and 101% in faeces).
Type:
other: Toxicokinetic evaluation of radioactivity levels in blood and plasma
Results:
After oral administration the measured radioactivity levels in blood and plasma were low, the majority of the measured values were below the lower limit of quantification. As a result, no reliable toxicokinetic evaluation could be performed.
Details on absorption:
Please refer to attached background material, which provides a Table with a summary of radioactivity data (Text Table 2).

The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose).

BILE-CANNULATED RATS (GROUP 5)
One animal (animal 18) died within 24 hours, this animal was excluded from the radioactive concentration calculations in the bile-cannulated group. Animal R3 showed a different radioactivity profile compared to the other two animals in the bile-cannulated group; higher total remaining amount in the carcass and lower amount in the feces. To visualize the difference between animal R3 and the other animals, the average value was calculated with and without animal R3. These values are presented between brackets below and in Text Table 2 in the attached background document.
After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 81% (96%) after 48 hours. Excretion via urine and bile were both minor routes of excretion, i.e. 0.03% (0.03%) for urine and <0.01% (<0.01%) for bile after 48 hours. These results indicate that the radioactivity excreted in the feces after oral administration consists of unabsorbed compound. In the bile-cannulated group, the average total remaining radioactivity in carcass inclusive blood and tissues was 15.9% (3.5%). Average total recovery of radioactivity after a single oral administration was 97% (100%) in the bile-cannulated group.
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 16% (4%). The relative high remaining radioactivity in the carcass and as a result the high oral absorption was caused by animal R3, where the total remaining amount in the carcass was 40.7%. This is likely due to radioactivity still being present in the GI tract, as the radioactivity in feces was much lower compared to the other two animals (49.8%). The radioactivity excreted via urine and bile was very low for this animal, comparable to the other two animals, therefore this radioactivity remaining in the GI tract is considered unabsorbed.



Details on distribution in tissues:
Not applicable.
Details on excretion:
Please refer to attached background material, which provides a Table with a summary of radioactivity data (Text Table 2).

After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Average total recovery of radioactivity after a single oral administration was 109% and 102% after repeated administration.

BILE-CANNULATED RATS (GROUP 5)
After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 81% (96%) after 48 hours. Excretion via urine and bile were both minor routes of excretion, i.e. 0.03% (0.03%) for urine and <0.01% (<0.01%) for bile after 48 hours. These results indicate that the radioactivity excreted in the feces after oral administration consists of unabsorbed compound. In the bile-cannulated group, the average total remaining radioactivity in carcass inclusive blood and tissues was 15.9% (3.5%). Average total recovery of radioactivity after a single oral administration was 97% (100%) in the bile-cannulated group.
Metabolites identified:
not measured
Details on metabolites:
Not applicable.
Bioaccessibility testing results:
Not applicable.

CLINICAL OBSERVATIONS

No mortality occurred, except for one animal (no. 18) in the bile-cannulated group that was found dead approximately 24 hours after radioactive dose administration. Piloerection and no feces production were observed. No macroscopic findings were observed at necropsy. This animal was excluded from the radioactive concentration calculations in the bile-cannulated group.

Animal R3 in the bile-cannulated group showed decreased food consumption and feces production compared to the other bile-cannulated animals, possible indicating a deteriorated health state of animal R3. Piloerection was noted after radioactive dose administration in almost all animals for a maximum of seven days. In addition, salivation, red discoloration of the feces and chromodacryorrhoea and rales were noted in the repeated dose groups after radioactive dose administration.

Table: Summary Radioactivity

Groups

Radioactivity

% of administered dose presented as mean ± SD

Urine

Feces

Bile

Total excretion

Carcass, incl. blood and tissues

Total recovery

Minimal oral absorption

Mass-balance

Group 1 - Single Dose

0.150±0.060

108.0±10.2

n/a

108.3±10.3

0.328±0.041

108.6±10.2

0.663±0.057

Mass-balance

Group 2 - Repeated Dose

0.148±0.052

100.8±4.24

n/a

101.1±4.15

0.406±0.099

101.5±4.11

0.748±0.242

Bile-cannulated

Group 51

0.033±0.009

(0.028)

80.7±26.9

(96.2)

0.006±0.001

(0.007)

80.7±26.9

(96.2)

15.9±21.5

(3.53)

96.7±5.41

(99.7)

16.0±21.5

(3.56)

1:            Values between brackets in Group 5 are the mean values without animal R3, as explained below in the bile-cannulated section of the summary.

n/a:       not applicable

Conclusions:
After oral administration at 1000 mg/kg bw of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Excretion via bile in bile-cannulated rats was ≤0.007%. The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose). After oral administration the majority of the measured values were below the lower limit of quantification and as a result, no reliable toxicokinetic evaluation could be performed.
Executive summary:

Male Wistar rats (4 per group) received either a single or a repeated oral dose of 1000 mg/kg bw of of [octadecyl-1-14C]Tetrameen 2HT Main component. The duration of sample collection following dosing was 168 hours for the mass balance groups 1 (single dose) and 2 (repeated dose), 24 hours for the toxicokinetics groups 3 (single dose) and 4 (repeated dose) and 48 hours for the bile-cannulated group, which received a single dose.

After administration of the radiolabeled dose in the mass-balance groups, urine was collected in 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours intervals. Feces were collected in 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours intervals. Volatiles were collected in 0-24 and 24-48 hours intervals. Animals were euthanized at the end of the collection period and cage washings were collected. The carcass was stored for total 14C analysis. Total radioactivity in urine, feces, volatiles, cage washings and carcass was determined.

In the toxicokinetic groups, blood was sampled from the jugular vein at pre-dose (Group 4 only) and 1, 2, 4, 7 and 24 hours after the radiolabeled dosing. Total radioactivity concentrations were determined.

In the bile-cannulated group, urine and bile were collected in 0-6, 6-24 and 24-48 hours intervals and feces were collected in 0-24 and 24-48 hours intervals. Animals were euthanized at the end of the collection period and cage washings were collected. The carcass was stored for total14C analysis. Total radioactivity in urine, bile, feces, cage washings and carcass was determined.

After oral administration at 1000 mg/kg bw of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Excretion via bile in bile-cannulated rats was ≤0.007%. The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose). After oral administration the majority of the measured values in blood and plasma were below the lower limit of quantification and as a result, no reliable toxicokinetic evaluation could be performed.

In conclusion, [octadecyl-1-14C]Tetrameen 2HT Main component administered orally was barely absorbed, not quantifiable in plasma and blood and mainly excreted unabsorbed via the feces.

(See graph cumulative excretion)

  • The repeated dose group shows no differences to single dose group.
  • The low inter-animal variability, including single & repeated dose groups, supports that these absorption characteristics are mostly caused by physico-chemical properties with little biological influence, suggesting also a low intra-species variability.
  • Considering radio-chemical purity of 93.4%, it is most likely that all this information is applicable to both the a.i as the impurities (conservative approach, as likely the impurities will be lower mw substances and likely to have a somewhat higher bioavailability)
  • The bile data shows that there is no entero-hepatic circulation taking place
  • The apparently higher absorption of bile group relates to higher carcass levels. Considering lower levels in urine it is actually indicating a lower level of systemic absorption. This means that carcass levels relate to unabsorbed remnants in intestines. Furthermore, bile group is only different to other with the evaluation of excretion via bile. They cannot be expected to show otherwise different absorption as non-surgically treated animals. (Besides, bile suggested to increase the uptake of fatty substances; bile cannulated animals do not produce bile for GI tract anymore, and if anything, absorption would possibly be even lower.)

Description of key information

Molecular profiling (with mw almost 700, Pow > 12, water sol. < 30 µg/L) suggests very low absorption.

This was confirmed by ADME testing according to OECD 417, which resulted to a calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing of on average 0.7% (single dose) and 0.8% (repeated dose). To this can be added that considering the radiochemical purity of 93.4%, it is most likely that this information is applicable to both the a.i as the impurities (conservative approach for AN-6, as likely the impurities will be lower mw substances and likely to have a somewhat higher bioavailability).

Due to lack of comparative quantitative data, absorption rates of 100% are indicated for all three routes. This basically indicates that in the considerations of DNEL setting, although the absorption is probably low, there is no significant difference taking into account in the comparison of absorption via dermal and inhalation routes compared to the oral route by which the hazard evaluations are done. Available studies do not indicate a concern for bioaccumulation.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

di-C16-C18 (evennumbered) alkyl tripropylenetetramine is a fatty amine (long alkyl chains linked to a nitrogen), linked to multiple (2 or more) 1,3-diamine propane (DP) groups. The mode of action for di-C16-C18 (evennumbered) alkyl tripropylenetetramine follows from its structure, consisting of an apolar fatty acid chain and a polar end of a primary amine.

The substance is completely protonated under environmental conditions which causes them to strongly adsorb to organic matter and to negatively charged cell membranes. The long apolar fatty acid chains easily dissolve in the cytoplasmic membrane whereas its function and structure is disrupted by the polar amine groups. The disruption of the cytoplasmic membrane causes cell damage or lyses of the cell content.Whether toxicological effects occur depends on whether local concentrations reach a level at which cellular integrity cannot be maintained. However, the whole molecule will not easily pass the membrane structure. The fraction of the quats adsorbed does not easily get free in solution again and the substance bound in the stratum corneum is not mobile.

Its molecular chemical profile and estimated properties is listed at a table below.

 

Absorption:

As this substance has a positive charge under physiological conditions (+4), transport across biological membranes is most likely further reduced which again would reduce oral absorption and bioavailability. This is confirmed inADME testing according to OECD 417, which resulted to a calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing of on average 0.7% (single dose) and 0.8% (repeated dose). To this can be added that considering the radiochemical purity of 93.4%, it is most likely that this information is applicable to both the a.i as the impurities (conservative approach, as likely the impurities will be lower mw substances and likely to have a somewhat higher bioavailability).

Due to lack of comparative quantitative datafor absorption over different routes, the absorption level is set at 100% for all routes. 

 

At this stage no data are available on dermal absorption. It is not expected to easily pass the skin in view of its ionised (4+) form under physiological conditions.Although dermal absorption is not expected at any considerable rate, also the absorption via dermal out is set at 100% as conservative approach(to indicate a similar absorption rate as via oral route).

 

Also for inhalationno data are available on absorption.With a very low vapour pressure (1.3 x 10-6Pa at 20°C; probably from impurities as calculated levels of 7.51 x 10-15Pa at 25°C from EPIWIN are even much lower), the potential for inhalation is limited. Relevant (in view of possible systemic absorption) exposures are only possible as aerosol. If any inhalation from aerosols does occur, this can only be in the form of larger droplets, as the use does not include fine spraying. Droplets will deposit mainly on upper airways, and will be subsequently swallowed following mucociliary transportation to pharynx. This results to no principal difference in absorption compared oral route. Absorption via respiratory route is therefore as worst case set at 100% (to indicate a similar rate as via oral route).

 

Distribution and excretion

Information from other cationic amines, including comparable monoalkyl polyamines, show very low oral absorption,low tissue distribution with most of the substanceprimarily associated with the intestinal mucosa, and excretion of any absorbed material mostly via urine.

ADME testing according to OECD 417 showed insignificant absorption: Total absorption was≤0.8% and showed vary low variability between animals and between single and repeated dose. Excretion as volatiles and in urine were both below 0.2%. Slightly higher levels of 0.3-0.4% were found in carcass (including blood and tissues), but likely this mostlyrelate to unabsorbed remnants in intestines.

Repeated dose studies have not shown accumulation or increased toxicity upon elongation of study duration.Low bioaccumulation potential fordi-C16-C18 (evennumbered) alkyl tripropylenetetramineis also shown by the low calculated BCF (3.162 L/kg) and low acute-to-chronic ratio of toxicity to aquatic species.

 

Metabolism

There is no specific information on metabolism ofdi-C16-C18 (evennumbered) alkyl tripropylenetetramine. In view of available information on various other alkyl amines, metabolism is expected to be very limited. In case of metabolism,oxidation of alkyl chains is expected via a process similar to beta-oxidation of fatty acids. The volatiles (0.2%) measured in the ADME study are more likely coming from radioactivity linked breakdown products.

 

Toxicological profile:

The acute oral toxicity of di-C16-C18 (evennumbered) alkyl tripropylenetetramine indicates a low acute toxicity with an LD50 cut-off of > 5000 mg/kg bw.

Potential exposure to di-C16-C18 (evennumbered) alkyl tripropylenetetramine is mainly dermal. As the substance is classified for severe skin irritation, effects following dermal exposures will be characterized by local skin effects that are related to duration, quantity and concentration of the substance, rather than by systemic toxicity due to dermal uptake. Especially considering that dermal uptake can be expected to be very limited.

Also no information on toxicity via inhalation route is available. As the substance is a viscous fluid with a high boiling point (> 400 °C) and very low vapour pressure (1.3 x 10-6 Pa at 20°C) inhalation possibility is limited and is only possible as aerosol. Furthermore, the substance is classified as severe irritant, whereas repeated dose studies by oral route indicated low systemic toxicity. This means that effects by inhalation will rather be characterised by local irritation than systemic toxicity.

 

The molecular structure of di-C16-C18 (evennumbered) alkyl tripropylenetetramine does not contain toxicophores indicating a concern for sensitization (See table with molecular profiling below), and cross-reading to data from structurally related substances also do not show a concern. Related to the properties of the substance (in principle a UVCB, mw almost 700, Pow > 12, water sol. < 30 µg/L) the substance is not suitable for testing in the currently accepted in vitro test systems. The only suitable in vitro system identified, SENS-IS, confirmed the non-sensitising properties. Although the substance was shown to be severely irritating to rabbit skin, SENS-IS also indicated non-irritating properties. This supports the notion that its high irritation to skin in in vivo studies is related to direct inflammatory reactions, an aspect which possibly complicates the evaluation for sensitising properties. The only available test system that in the assessment for sensitisation includes evaluation elicitation phase involves the guinea pig studies such as the GPMT. Considering that the properties of Di-C16-C18 (evennumbered) alkyl tripropylenetetramine results in an expected low dermal bioavailability (mw almost 700, Pow > 12, not soluble in water), the GPMT involving intradermal injection of test substance together with FCA to enhance sensitisation reactions, can be considered as the most stringent test system possible for the evaluation of this substance. This study shows some inflammatory effects, but not sufficient to indicated increased inflammatory reactions following the process of induction.

 

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine also has no genotoxic properties. The substance was not mutagenic in a bacterial mutagenicity study (Ames test), and evaluated negative in an in vitro micronucleus assay in cultured peripheral human lymphocytes (OECD 487). Also results from molecular profiling does no give rise for concerns for genotoxicity.

 

The available data from the combined 28-day/reproduction screening (OECD 422) study in rats involving exposures up to 54 days by gavage, did not show significant effects up to highest dose of 1000 mg/kg bw. A conservative NOAEL of 100 mg/kg was selected based on an increase of macrophage foci in mesenteric lymph nodes and macrophage foam cells in the lamina propia of small intestines.

In a combined 90-day/2-generation study with dosing via diet, toxicity was observed at the higher dose groups, considered to be mediated by malnutrition from interaction with food. The study resulted to a NOAEL < 1000 ppm (72 – 90 mg/kg bw/day in males and females over both generations), based on minimal to slight necrotizing granulomas in mesenteric lymph nodes observed in some animals at the lowest dose group of 1000 ppm.

Extrapolation of the observed effects in mesenteric lymph nodes indicates a NOAEL of around 20 mg/kg bw/day (conservative, as extrapolation of results from OECD 422, and of F0 and F1 results from 90-day/2-generation, indicates a NOEL between 28 and 52 mg/kg bw). This would represent a threshold for the effects irrespective the duration of the study (45 day in OECD 422 and 90 up to 126-days in the 2-generation study). Although the threshold for effects seems the same, with increasing duration the severity of effects at higher dose levels tend to increase.

 

Both the combined 28-day/reproduction screening (OECD 422) and the 90-day/2-generation study basically indicate no specific concerns for reproduction toxicity. No effects were seen in the OECD 422 study at the highest test dose of 1000 mg/kg bw/day for maternal toxicity, development and reproduction endpoints. Also, no developmental toxicity was observed in an OECD 414 developmental toxicity study in rats when tested up to 1000 mg/kg bw/day.

The combined 90-day/2-generation study with dosing by diet rather than gavage resulted to health effects from severe malnutrition at the highest dose level of 10 000 ppm (corresponding to 930 to 1010 mg/kg bw/day), causing secondary effects on fertility following acyclicity of the females.

At 3000 ppm (corresponding to 245 to 309 mg/kg bw/day) areduced pup body weight and reduced body weight gain was observed in both F1 and F2 generations. The lower BW is comparable for F0, F1 and F2 and remains the same over all dosing periods, so it is not considered to be a specific developmental effect.

 

In view of the limited uptake, and general low toxicity of the substance, no effects on or via lactation are to be expected.

  

Molecular chemical profile and estimated properties:

 

di-C16-C18 (evennumbered) alkyl tripropylenetetramine (below based on 2C18)

Chemical name

di-C16-C18 alkyl (evennumbered) tripropylenetetramine

[3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dioctadecylamine

CAS

1623405-26-4

Physical state

Viscous liquid (borderline of paste/viscous liquid)

SMILES

CCCCCCCCCCCCCCCCCCN(CCCCCCCCCCCCCCCCCC)CCCNCCCNCCCN

Molecular structure

(HT-alkyl)2-N-CH2CH2CH2-N-CH2CH2CH2-N-CH2CH2CH2-N

(In SMILES structure HT is represented as C18)

Molecular formula

C45H96N4

Molecular weight

693,2705 (2C18 = 692; 2C16 = 636)

Solubility: avgLogS

WSKOW v1.42

WATERNT v1.01

2.55 µg/L (ALOGpS: 18.61 µg/L; AC logS: 0.35 µg/L)

5.777e-012 mg/L (25 °C)

6.9329e-007 mg/L (25 °C)

Solubility (meas)

Insoluble in (cold) water

Density (20°C)

0.86 g/mL

pKa: (4 nitrogens)

pKa's 11.19, 7.00, 10.40, 8.57 (in structure from left to right)

4+: > 99% at pH ≤ 5.00

Neutral: <1% at pH < 9.8

logPow   (ALOGPS 2.1)

(KOWWIN v1.68)

12.80 (± 3.44)

16.2014

logPow (meas)

No measured log Kow value was derived because of the extreme low water solubility and high predicted log Kow. For very hydrophobic substances there are too many experimental difficulties to obtain values with sufficient certainty. For substances with a calculated log Kow higher than 10 the REACH PBT guidance R.11 states that the substances BCF is probably lower than 2000. This is confirmed by the low calculated log BCF values for di-C16-C18 (evennumbered) alkyl tripropylenetetramine resulting to 3.16 L/kg ww.

logD (Chemaxon)

pH       logD

1,700   0,925

4,600   1,882

5,500   3,029

7,000   5,761

mp (EPIWIN)

310.67 °C

bp (EPIWIN)

709.95 °C

Vp (EPIWIN)

7.51E-015 Pa at 25°C

mp (measured)

17,5 ºC

bp (measured)

> 400 ºC

vp (measured)

< 0.002 Pa at 20°C (based on RA di-C16-C18 alkylamine)

Reactivity

Alerts:

- DNA binding by OECD: Structural alert: Aliphatic tertiary amines

[P450 metabolism to a reactive iminium species has been suggested as a potential pathway to DNA adducts via an SN1 mechanism]

This alert is triggered for all tertiary amines, including the polyamines. However, none of the many available studies for these substances have shown any concern.

- Repeated dose (HESS): Mucous membrane irritation (Rank C)

- Toxic hazard classification by Cramer: High (Class III): based on: Containing an aliphatic secondary amine

No alerts for:

Mechanistic grouping

- DPRA Cysteine/ Lysine peptide depletion: outside domain

- Estrogen Receptor Binding: no binder

- Protein binding by OASIS v1.3: No alert found

- Protein binding by OECD: No alert found

- Protein binding potency: outside domain

- DNA binding by OASIS v1.3: No alert found

Endpoint specific

- Carcinogenicity (genotox and nongenotox) alerts by ISS: No alert found

- DART scheme v.1.0: none

- DNA alerts for AMES, MN and CA by OASIS v.1.3: No alert found

- in vitro mutagenicity (Ames test) alerts by ISS: No alert found

- in vivo mutagenicity (Micronucleus) alerts by ISS: No alert found

- Oncologic Primary Classification: No alert found

- Protein binding alerts for Chromosomal aberration by OASIS v1.1: No alert found

- rtER Expert System ver.1 – USEPA: No alert found

Dermal penetration coefficient Kp (est)

1.18e+004 cm/hr (water:0.0005 cm/hr)

Human Intestinal

absorption (HIA)

65.3%

Lipinski's rule of five: no

Bioavailability: no

ADMET: Very Poor absorption

Molecular formula, molecular weight, pKa and logD were all calculated using ChemAxon MarvinSketch (v.6.1.6).

Melting point, boiling point, vapour pressure and logPow were estimated by EPI Suite (v4.1).

Solubility and logPow are estimated using ALOGPS 2.1 (VCCLAB, Virtual Computational Chemistry Laboratory,http://www.vcclab.org, 2005)

Reactivity: QSAR Toolbox v.3.3.

Absorption properties:

- dermal: EpiSuite v. 4.1; (water:0.0005 cm/hr): log Kp = -2.80 + 0.66 log Kow - 0.0056 MW

- intestinal: HIA: QSAR toolbox (version 3.2) (Human Intestinal Absorption)