Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

distribution

Qualifier:

equivalent or similar to guideline

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 or test system 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.

Dose / conc.:

6.25 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose / concentration:

6 for low dose
9 for high dose

Control animals:

no

Positive control reference chemical:

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:

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 of ¹⁴C-Octadecyl 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 radiolabelled 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.

Description of key information

There are no specific studies available on absorption, distribution, metabolism or excretion of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates.

Key value for chemical safety assessment

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

When diamines acetates (DoAc) dissolve in water or physiological media, the solid structure of the salt is broken up by water molecules surrounding the charged particles namely, the positive diamine and negative acetate ions. Diamines are strong bases with a pKa of about 10.6. Therefore, the amines under physiological conditions will be protonated largely. Although, the solubility would be increased and the pH neutralised in part for the DoAc, it is expected that its behaviour will not be far different from that of the diamines as described below with the data of N-(hydrogenated tallow alkyl) trimethylenediamine, CAS 68603-64-5 (recently redefined as N-C16-18-alkyl (even numbered)-1,3-diaminepropane, CAS 133779-11-0), also referred to as HT-diamine:

 

Physical-chemical properties

HT-diamine has a molecular weight range of 298 - 327 g/mol (avg. 319 g/mol) and is a white amorphous solid. The substance has a measured melting point of 44°C, a measured boiling point of > 250°C at 1013 hPa and a measured vapour pressure of 0.0015 Pa at 20°C (worst case based on read-across from shorter chain C_12-14-diamine). The octanol-water partition coefficient (log Pow) is 1.5 at 25.7 °C and the water solubility (expressed as the Critical Micelle Concentration or CMC) regarding to an analogue compound (C18-unsatd diamine) at 36 mg/L at pH 7 and 25°C.

In physiological circumstances are both nitrogens positively charged, resulting to 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. Consequently, the whole molecule will not easily pass membrane structures. Cytotoxicity at the local site of contact through disruption of cell membrane is considered the most prominent mechanism of action for toxic effects.

 

Absorption (oral), distribution, metabolism, excretion

A well performed study (Active Biotech research, 2009) with N-C16-18-alkyl (even numbered)-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 of ¹⁴C-Octadecyl-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 radiolabelled 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 the lack of quantitative absorption data and in accordance with ECHA, the oral absorption is assumed to represent 50% of the absorption following the inhalation of the substance. Since the inhalation absorption is assumed to be 100% as it represents a worst-case scenario in the absence of experimental data, the oral absorption is considered to be 50%.

 

 

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. Based on the corrosive properties of most di-amines, and the highly irritating properties of the others (as well as diamines acetates), dermal absorption because of facilitated penetration through damaged skin can be anticipated. Dependent on the solvent and concentration, up to 60% dermal absorption may be taken as a worst case for assessment purposes (value taken from the existing EU risk assessment on primary alkylamines).

Due to the lack of quantitative absorption data and in accordance with ECHA, the dermal absorption is assumed to represent 50% of the absorption following the inhalation of the substance.

 

 

Inhalation:

N-C16-18-alkyl (even numbered)-1,3-diaminepropane is a solid (pellets) with a melting point of 43°C, with no inhalable particles (0.11% (m/m) with particle size < 100µm) and is marketed as a viscous liquid 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 C_12-14-diamine). In addition, 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.

Due to the lack of quantitative absorption data, a 100% absorption is taken as a conservative approach.