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Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near-guideline study acceptable for assessment.
Justification for type of information:
Further information on the applicability of the read-across from various diamines to C12/14-diamine can be obtained from the document "Category polyamines - 20170314.pdf" added to IUCLID Ch. 13.
Objective of study:
distribution
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Principles of method if other than guideline:
The rats were subjected for whole body autoradiography and sectioned sagittaly according to the standard method, (Ullberg 1977 and Ullberg et al 1982).
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C-Octadecyl diamine
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Species: Rat
Strains: Sprague-Dawley
Supplier: Taconic, Denmark
Weight 150 g on arrival

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dosing Sex Route
of adm. Dose Survival time
after admin.
µCi/kg
mg/kg
Period 1,
Rep, (6xcold,1 hot) low dose M p.o. 250/6.25 1h
Rep, (6xcold,1 hot) low dose M p.o. 250/6.25 4h
Rep, (6xcold,1 hot) low dose M p.o. 250/6.25 24h
Rep, (6xcold,1 hot) high dose M p.o. 250/62.5 1h
Rep, (6xcold,1 hot) high dose M p.o. 250/62.5 4h
Rep, (6xcold,1 hot) high dose M p.o. 250/62.5 24h

Period 2,
Rep, (2xcold,1 hot) high dose M p.o. 250/62.5 1h
Rep, (2xcold,1 hot) high dose M p.o. 250/62.5 4h
Rep, (2xcold,1 hot) high dose M p.o 250/62.5 24h

Period 3,
Single, low dose M p.o. 250/6.25 1 h
Single, low dose M p.o. 250/6.25 4 h
Single, low dose M p.o. 250/6.25 24 h
Single, high dose M p.o. 250/62.5 1 h
Single, high dose M p.o. 250/62.5 4 h
Single, high dose M p.o. 250/62.5 24 h
Duration and frequency of treatment / exposure:
Single application in two dose levels and with 3 survival time points.
Two daily repeated applications in the high dose level with "cold" test item followed by one application of radiolabeled test item on 3rd day and with 3 survival time points.
Six daily repeated applications in two dose levels with "cold" test item followed by one application of radiolabeled test item on 7th day and with 3 survival time points.
Remarks:
Doses / Concentrations:
low dose: 6.25 mg/kg bw
high dose: 62.5 mg/kg bw
No. of animals per sex per dose:
6 for low dose
9 for high dose
Control animals:
no
Positive control:
no
Details on dosing and sampling:
The rats were anaesthetized by Sevoflurane, and then immediately immersed in heptane, cooled with dry ice to -70°C, according to ABR-SOP-0130. The frozen carcasses were embedded in a gel of aqueous carboxymethyl cellulose (CMC), frozen in ethanol, cooled with dry ice (-70° C) and sectioned sagittaly for whole body autoradiography, according to the standard method, (Ullberg et al 1982 and Ullberg 1977). From each animal 20 p.m sections were cut at different levels with a cryomicrotome (Leica CM 3600) at a temperature of about -20°C. The obtained sections were caught on tape (Minnesota Mining and Manufacturing Co., No. 810) and numbered consecutively with radioactive ink. After being freeze-dried at -20°C for about 24 hours, the sections were put on 14C-imaging plates (Fuji, Japan).

Sections were chosen for phosphor imaging (Amemiya Y et al 1987) to best represent the tissues and organs of interest. Together with a set of 4C calibration standards, the sections were then put on imaging plates. The imaging plates were exposed for 4-5 days enclosed in light tight cassettes at -20°C in a lead shielding box to protect from environmental radiation.
After exposure the imaging plates were scanned at a pixel size of 50 pm using BAS 2500 (Fuji Film Sverige AB, Sweden). The tissues and organs of interest were quantified using AIDA, version 4.19 (Raytest, Germany) (Ahr H.J. and Steinke W., 1994)
A water-soluble standard test solution of 14C-radioactivity was mixed with whole blood and used for the production of the calibration scale. The 14C calibration standards consisted of 10 dilutions from 289.5 to 3.559 kBq/g. For the purpose of quantification, it was assumed that all tissues had similar density and quench characteristics as that of whole blood. The tissue density was set to 1 g/mL. The limit of quantification was defined as the mean concentration value of eight measurements for background plus three times the standard deviation value of these measurements.
The various tissues and organs were identified either on the autoradiogram or on the corresponding tissue section.
Statistics:
Not applicable
Type:
distribution
Results:
Distribution was similar, independent of time points, doses or dose regimens. Highest tissue concentrations of radioactivity were registered in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal, myocardium and brown fat.
Type:
absorption
Results:
The labeled diamine seemed to be quite slowly absorbed from the gastrointestinal tract. The blood radioactivity was low and slightly above LOQ for the different doses and dose regimens.
Details on distribution in tissues:
Tissue distribution and tissue uptake
The test item showed quantifiable levels of radioactivity for most tissues at 4 and 24 hours but almost no radioactivity at 1hour.
The distribution pattern was similar for all animals independent of time points, doses or dose regimens.
The highest tissue concentrations of radioactivity were registered in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal, myocardium and brown fat.

Time dependence
The test item was slowly absorbed from the gastrointestinal tract, and blood radioactivity was low and slightly above LOQ.
The highest concentration of radioactivity was obtained at 24 hours for almost all tissues.

Dose dependence
Seven daily repeated applications of 6.25 mg/kg compared to corresponding daily applications of 62.5 mg/kg showed similar tissue to blood ratios at 4 hours.
At 24 hours the tissue to blood ratios were generally up to two times higher for the low dose compared to the high dose.
A single application of the low dose compared to the high showed similar tissue to blood ratios at 4 hours, but at 24 hours it showed two to three times higher tissue to blood ratios.

Single and repeated high dose dependence
Three daily applications of the test item compared to a single dose generally showed slightly higher tissue to blood ratios at both 4 and 24 hours.
Seven daily applications compared to a single application of the test item showed similar tissue to blood ratios at both 4 and 24 hours.
Seven daily applications of the test item compared to three, showed slightly lower tissue to blood ratios at both 4 and 24 hours.
Metabolites identified:
no
Conclusions:
Interpretation of results: bioaccumulation potential cannot be judged based on study results
Distribution was similar, independent of time points, doses or dose regimens. Highest tissue concentrations of radioactivity were registered in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal, myocardium and brown fat.The labeled diamine seemed to be quite slowly absorbed from the gastrointestinal tract. The blood radioactivity was low and slightly above LOQ for the different doses and dose regimens.
Executive summary:

The aim of the study was to determine tissue/organ distribution of14C-Octadecyl diamineafter oral administration in the male rat using quantitative whole-body autoradiography. Specifically: the rats were given 62.5 or 6.25 mg/kg body weight of the test item by gavage, and were killed 1, 4 and 24 hours after the last administration.

Two daily repeated applications in the high dose level with "cold" test item followed by one application of radiolabeled test item on the 3rd day.

Six daily repeated applications in two dose levels with "cold" test item followed by one application of radiolabeled test item on the 7th day.

Generally, the distribution pattern was similar for all animals independent of time points, doses or dose regimens. The highest tissue concentrations of radioactivity were registered in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal cortex, myocardium and brown fat. All other tissues showed low or very low levels of radioactivity.

 

The labeled diamine seemed to be quite slowly absorbed from the gastrointestinal tract. The blood radioactivity was low and slightly above LOQ for the different doses and dose regimens. Furthermore, for almost all tissues the highest concentration of radioactivity was obtained at 24 hours after the administration.

Description of key information

Due to lack of quantitative data, absorption rates of 100% are indicated for all three routes. This basically indicates that, although the absorption is probably low, it is considered that no significant difference in absorption occurs between oral, dermal and inhalation route. Very likely this means an overestimation of the dermal absorption compared to oral route. Available studies do not indicate a concern for bioaccumulation. An ADME study applying radio-labelled Hydrogenated tallow diamine by oral route indicates slow absorption, with highest concentrations of radioactivity seen in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal cortex, myocardium and brown fat. All other tissues showed low or very low levels of radioactivity.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

The hazard evaluation of (Z)-N-9-octadecenyl-1,3-diaminopropane (Oleyl diamine) includes highly reliable GLP studies performed over the category of alkyl-diamines. Cross-reading to data available on other diamines and polyamines is acceptable on the basis of identical alkyl-diamine structure, resulting to the same functional groups with similar properties leading to common biological activity, and common metabolic degradation. Further information on the applicability of the read-across from various diamines to Oleyl-diamine can be obtained from the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.

 

1. Physical-chemical properties

The test substance, (Z)-N-9-octadecenyl-1,3-diaminopropane (Oleyl diamine) CAS 7173-62-8, has a molecular weight of 324.67 g/mole and is at room temperature a white-grey liquid with solid parts as being in semi melted stage. The substance has a melting point of 3°C, a boiling point of > 150°C at 1013 hPa and a vapour pressure less than 0.0015 Pa at 20°C (worst case, as based on read-across from shorter chain C12-14-diamine). Solubility and Pow are pH dependent. The octanol-water partition coefficient (log Pow) is 0.0 at 25.7 °C and as the substance forms micelles in water the water solubility is expressed as the critical micelle concentration (CMC, a solubility limit) at 36 mg/L at pH 7 and 23°C.

In physiological circumstances, both nitrogens are positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces such as cell 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. Cytotoxicity at the local site of contact through disruption of cell membrane will is considered the most prominent mechanism of action for toxic effects.

 

2. Data from acute toxicity studies and irritation studies

Data more relevant for the classification for acute toxicity is derived from series of highly reliable GLP (Acute Toxic Class, OECD 423) studies performed together over the category of alkyl-diamines. LD50 for Oleyl diamine ranges between 500 and 1000 mg/kg bw.

Oleyl diamineis corrosive to the skin and is not expected to easily pass the skin in view of its ionised form at physiological conditions. Testing via dermal route for is therefore not a preferred route. However, as dermal absorption is not quantitatively evaluated, 100% is assumed as worst case assumption.

 

3. Data from repeated dose toxicity studies

Oral:

A 90-day study is available for N-C12-14 alkyl-1,3-diaminopropane (C12/14-diamine). Cross-reading to this substance is considered acceptable on the basis of similarities of structure with same functional groups and properties, leading to common biological activity and common kinetics and metabolic route of metabolism. The shorter alkyl chains of the C12/14-diamines, compared to the Oleyl diamine consisting of C18 alkyl chains, can be considered to be a worst case representation. This is supported by the results from repeated dose studies with Oleyl-diamine itself, showing an identical profile but higher dose levels, resulting to higher NOAEL.

 

N-C12,14 alkyl-1,3-diaminopropane was tested in Wistar rats in a 90-day repeated dose toxicity study at 0 (vehicle control), 0.1, 0.4, 1.5 or 6 mg/kg/day. At macroscopic examination no test item-related macroscopic organ lesions were seen.

Histopathologically, test item-related changes were observed in the small intestine (ileum and jejunum), mesenteric lymph node, spleen, bone marrow (sternum) and trachea.

In the small intestinal villi, minimal to moderate accumulations of foam cells, interpreted to represent foamy histiocytes, were seen in the jejunum and ileum, predominantly at 6 mg/kg/day, but also in one female at 1.5 mg/kg/day.

There was a dose-related foam cell infiltration in the mesenteric lymph node (draining lymph node of the small intestine), in rats treated at 1.5 and 6 mg/kg/day, associated with foci of necrosis/abscessation or of fibrosis in some animals treated at 6 mg/kg/day.

 

Small intestinal and mesenteric lymph node lesions were not reversible during the 28-day recovery period, but had partially regressed after the 90-day recovery period. Spleen, bone marrow and tracheal changes had completely regressed after the 28-day and 90-day recovery period.

 

The most significant treatment-related changes in all studies with cationic surfactants are the effects on the small intestine and mesenteric lymph nodes, which only partially regressed during the recovery period. A relatively strong inflammatory reaction is also observed at higher dose levels. These effects have consistently been observed with these substances, which support the current approach of grouping of substances and read-across of data.

 

Except histopathology, slight effect on body weight, food consumption and clinical signs, no test item related effect was observed in any of the parameters evaluated in mid and high dose groups in the 90-day study. Based on these findings and the histopathological evaluation, the NOAEL (No-Observed-Adverse-Effect-Level) was considered to be 0.4 mg/kg/day under the circumstances of the study.

 

The similarity of the findings in these studies (and various others performed on similar substances) supports the acceptability of grouping and the cross-reading between the diamines, and also indicated that the shorter chain diamines results to the lowest NOAEL. Using the data from C12-14-diamine thus represents a worst case approach in the risk assessment of the longer chain alkyl-diamines.

 

Also a 15-day study on Coco-diamine itself shows comparable results. The higher NOAEL level of 6 mg/kg bw/day however suggests that 14-day might just not be long enough for the effects to fully develop, whereas the NOAEL from the 28-day and the 90-day study do not differ. As the NOAEL does not seem to be influenced by the duration of the study beyond 28-days, this is indicative for the lack of bio-accumulating potential of the diamines.

 

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.

 

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.

Phospholipidosis is a plausible mechanism. In physiological circumstances, the diamines 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 all lysosomotropic, and scored positive for phospholipidosis in in vitro studies with HepG2 cells.

 

Inhalation:

Oleyl diamine is a liquid/paste with a vapour pressure less than 0.0015 Pa at 20°C (value is an overestimation as it is based on read-across from shorter chain C12-14-diamine). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur.

 

Dermal:

No data from repeated dose studies via dermal route. Oleyl diamine is corrosive to the skin.The diamines are on overall corrosive to the skin. For these corrosive effects to the skin, a trend can be observed, with higher corrosion for shorter chain lengths (C12-14-diamine vs C16-18-diamine) and for higher levels of unsaturation (Oley-diamine vs HT-diamine). This could be related to the physical state of the substance: The more liquid the better the contact to the skin and penetration to the viable cell layers.

Diamines are not expected to easily pass the skin in view of their ionised form at physiological conditions. The dermal route is therefore not a preferred route for dosing when evaluating repeated dose toxicity. In addition, there is no consumer exposure to HT-diamine. Further, manufacture and use are highly controlled. Its use is limited to industrial and professional users where following its severe corrosive properties the applied protection measures will provide for sufficient protection to prevent exposure.

 

3. Absorption, distribution, metabolism, excretion

A well performed study (Active Biotech research, 2009) withN-C16-18-alkyl (evennumbered)-1,3-diaminepropane with14C-labelled C18-diamine, gives some information on the tissue distribution of the test item that is required to help predict its efficacy and the duration of its effect and to provide supportive data on the mechanism of results from laboratory animals. The aim of the study was to determine tissue/organ distribution of14C-Octadecyl-diamine (C18-diamine) after oral administration in the male rat using quantitative whole-body autoradiography. Specifically: the rats were given 62.5 or 6.25 mg/kg body weight of the test item by gavage, and were killed 1, 4 and 24 hours after the last administration.

Two daily repeated applications in the high dose level with "cold" test item followed by one application of radiolabelled test item on the 3rd day.

Six daily repeated applications in two dose levels with "cold" test item followed by one application of radiolabeled test item on the 7th day.

Generally, the distribution pattern was similar for all animals independent of time points, doses or dose regimens. The highest tissue concentrations of radioactivity were registered in the intestinal mucosa, abdominal lymph nodes, liver, spleen, adrenal cortex, myocardium and brown fat. All other tissues showed low or very low levels of radioactivity.

 

The labelled diamine seemed to be quite slowly absorbed from the gastrointestinal tract. The blood radioactivity was low and slightly above LOQ for the different doses and dose regimens. Furthermore, for almost all tissues the highest concentration of radioactivity was obtained at 24 hours after the administration. However, due to lack of quantitative data, the absorption rate following oral dosing is considered to be 100%.

 

The findings in the toxicokinetics study are in agreement with the toxicological results from the repeated dosing studies by oral gavage. Octadecyl diamines when administered orally are not extensively absorbed, probably due to its low solubility and CMC formation. The effects of the diamines on which the NOAEL’s are based in the 28 and 90-day repeated dose toxicity studies, effects in the small intestinal and mesenteric lymph node lesions intestines, indicate local effects, and can probably be considered local NOAEL’s.

 

 

Dermal absorption

At this stage no data are available on dermal absorption. In physiological circumstances, both nitrogens are positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces such as cell 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 through membrane structures.

Based on the corrosive properties of most di-amines, facilitated dermal absorption as a consequence of facilitated penetration through damaged skin can be anticipated. The existing EU risk assessment on primary alkylamines considered that absorption could be dependent on the solvent and concentration, and decided that up to 60% dermal absorption may be taken as a worst case for assessment purposes.

On the other hand however, there is information that these substances adsorb into the str. corneum, but do not penetrate skin. Data fromin vitrotesting on comparable cationic surfactants show that therateof absorption is extremely low.

Due to the lack of quantitative absorption data for comparison between oral and dermal absorption, 100% absorption is taken as a conservative approach.