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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles.
Objective of study:
absorption
Principles of method if other than guideline:
The absorbability of the fatty acid moiety of the complete, oleate esters of alcohols containing from one to six hydroxyl groups was determined by the fat balance technique in adult rats. Similarly, the absorbability of sucrose octaoleate and sucrose monooleate was determined.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: no data
- Age at study initiation: young adult
- Weight at study initiation: approx. 200 g
- Housing: Individually in cages with raised screen bottoms
- Diet (e.g. ad libitum): ad libitum
Route of administration:
oral: feed
Duration and frequency of treatment / exposure:
10 Days, diet ad libitum
Remarks:
Doses / Concentrations:
10% and 25 % of dietary fat
Details on absorption:
The fatty acids of the compounds containing less than four ester groups, methyl oleate, ethylene glycol dioleate, glycerol trioleate, and sucrose monooleate, were almost completely absorbed. As the number of ester groups was increased - erythritol and pentaerythritol tetraoleate and xylitol pentaoleate - the absorbability decreased. The fatty acids of sorbitol hexaoleate and sucrose octaoleate were not absorbed. These differences in absorbability are related to the activity and specificity of the lipolytic enzymes in the lumen of the intestinal tract.

The fatty acids of the compounds containing less than four ester groups, methyl oleate, ethylene glycol dioleate, glycerol trioleate, and sucrose monooleate, were almost completely absorbed. As the number of ester groups was increased - erythritol and pentaerythritol tetraoleate and xylitol pentaoleate - the absorbability decreased. The fatty acids of sorbitol hexaoleate and sucrose octaoleate were not absorbed. These differences in absorbability are related to the activity and specificity of the lipolytic enzymes in the lumen of the intestinal tract.

Test fat

Percentage of dietary fat

Absorbability [%]

Methyl Oleate

10

100

25

96

Ethylen Glycol Oleate

10

100

25

92

Glycerol Trioleate

100

(100)

Erythritol Tetraoleate

10

-

25

72

Pentaerythritol Tetraoleate

10

90

25

64

Xylol Pentaoleate

10

50

25

24

Sorbitol hexaoleate

10

0

25

0

Sucrose Octaoleate

5

0

10

0

15

0

Sucrose Monooleate

5

100

10

100

15

100

Conclusions:
Absorption rates were between 0 an 100 %, depending on the amount of ester groups present in the substance fed. Pentaerythritole tetraoleate had an absorption rate of 90% (10% of dietary fat) and 64% (25% of dietary fat), respectively. Erythritole tetraoleate had an absorption rate of 72% (25% of dietary fat).
Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
Principles of method if other than guideline:
The mechanism of the intestinal fat absorption has been studied with 14C labeled fat in rats with the intestinal lymph duct cannulated.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C labeled fat
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: gavage
Duration and frequency of treatment / exposure:
single oral exposure
(at least 18 hours after surgery)
Remarks:
Doses / Concentrations:
A) 0.5 mL corn oil + 2.5 mg active palmitic acid-1-14C
B) 0.5 mL corn oil transesterified with 2.5 mg active palmitic acid-1-14C
C) 0.5 mL hydrolysed corn oil + 2.5 mg active palmitic acid-1-14C
No. of animals per sex per dose:
5-6
Control animals:
no
Details on absorption:
24 hours after administration of the different fats the mean recovered activities in lymph were as following:
A) 0.5 mL corn oil + 2.5 mg active palmitic acid-1-14C: 57.0 %
B) 0.5 mL corn oil transesterified with 2.5 mg active palmitic acid-1-14C: 61.7 %
C) 0.5 mL hydrolysed corn oil + 2.5 mg active palmitic acid-1-14C: 62.3 %

In all three groups of experiments maximum recoveries were found after 24 hours, i.e. 80.9, 85.0 and 87.5 % of the activity given.
Free fatty acids administered alone or together with glycerides appear in the lymph in glycerides and phospholipids.
No free fatty acids or soaps appear in the lymph.
The intestinal wall supplies a quantitatively important part of phospholipids to the blood during fat absorption.
The recoveries in the lymph of the fat fed varied widely. Diarrhea occured in some animals especially after feeding hydrolysed corn oil.
Details on distribution in tissues:
Absorbed fat is mainly transported via lymphatic channels to the systemic circulation whether fed as glycerides or as fatty acids.
Details on metabolites:
A complete hydrolysis of the fat in the intestinal lumen might occur in the rat.

The proportions of neutral fat and phospholipids in the lymph were in all three cases about the same. 90% of the fatty acids were present in the neutral fat and the remaining 10 % in phospholipids. The neutral fat consisted chiefly of triglycerides; cholesterol and cholesterol esters representing only a minor part of this fraction. No free fatty acids or soaps appeared in the lymph.

The results indicated that glycerides might be completely hydrolysed in the intestinal lumen of the rat and then resynthesized in the intestinal wall.

Conclusions:
Mean absorption rate of corn oil combined with palmitic acid was between 57 - 62 %.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles
Objective of study:
metabolism
Principles of method if other than guideline:
The lipolytic activity of human gastric and duodenal juice against medium chain and long chain triglycerides was compared.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
Glyceryl trioctanoate-1-14C
Species:
human
Route of administration:
other: in vitro testing

Enzymatic Lipolysis by Gastric and Duodenal Juice:

All samples of gastric juice showed lipolytic activity against trioctanoin and triolein. Hydrolysis of emulsified trioctanoin was greater than of emulsified triolein. Hydrolysis of unemulsified trioctanoin was less and more variable.

Duodenal juice was more active, even against unemulsified trioctanoin and triolein. Duodenal juice was more active against unemulsified substrate than gastric juice against emulsified substrate.

Table 1: Hydrolysis of trioctanoin and triolein*

 

Substrate and form

(μmoles)

Hydrolysis (%)

 

Trioctanoin

Triolein

Gastric juice

30, unemulsified

21

1

 

60, emulsified

33

16

Duodenal juice

30, unemulsified

40

34

 

45, emulsified

42

35

 

105, emulsified

45

36

*Gastric or duodenal juice (1 mL) was incubated (1 hour, continuous shaking, 37ºC) with 1 mL of buffer and unemulsified substrate or 1 mL of substrate emulsified in 10 mM sodium taurodeoxycholate, pH6.

pH Optimum

In the presence of bile acids, gastric lipolytic activity against trioctanoin had a broad pH optimum, between 4 and 7. The lipolytic activity of duodenal juice had a sharper pH optimum, between 6 and 8. The pH optimum was lower for short chain triglycerides, indicating that pH optimum values for lipases must be defined for a particular substrate.

Chain Length Specificity

Lipolysis rates increased with decreasing chain lengths for pure triglycerides.

Tributyrin was cleaved more rapidly than trihexanoin which in turn was cleaved more rapidly than trioctanoin (ratio of rates, 100:69:53). Because the pH optimum of gastric lipase is lower for short chain triglycerides than for MCT, trihexanoin and tributyrin were cleaved much more rapidly than, for example, trioctanoin at pH5.

Esterification and Fatty Acid Acceptors by Gastric and Duodenal Lipases

Gastric and duodenal lipases did not induce esterification of the fatty acid acceptor, glyceryl 2 -monooleyl ester, by octanoic acid over the pH range of 2 to 6. However, it was esterified by oleic acid in the presence of gastric juice, duodenal juice, or pancreatic fistula juice when bile acids were added. Esterification, calculated by disappearance of titratable fatty acid, was confirmed by TLC which showed the formation of compounds having the mobilities of a monoether monoester and a monoether diester. Control incubations without enzyme showed no loss of oleic acid or appearance of new lipids by TLC. To determine the amount of disubstituted and trisubstituted glyceryl derivatives which were formed, 14C-labeled glyceryl 2 -monooleyl ether was used and the products of the reaction were examined by zonal scanning. The glyceryl 2 -monooleyl ether was not cleaved during the incubation procedure. The amounts of ester bonds formed estimated by titration and by zonal scanning were in good agreement.

Products of Lipolysis and Positional Specificity

The specificity of pancreatic lipase for the 1 -ester bond in LCT has been demonstrated previously by establishing the formation of 2 -monoglycerides and fatty acid as end products of lipolysis. This procedure cannot be used for MCT because medium chain 2 -monoglycerides are either cleaved by pancreatic lipase or rapidly isomerized to the 1 -isomer which is rapidly hydrolyzed or both. Indeed, chromatographic examination of the products of hydrolysis of trioctanoin-14C showed only a small fraction of monoglyceride present.

Table 2: Products of hydrolysis of trioctanoin by gastric juice*

 

Radioactivity distribution** (%)

Lipolysis

(%)

 

Monoglyceride

Diglyceride

Fatty acid

Triglyceride

Buffer (control)

0

0

0

100

0

Gastric juice

1 mL

3

26

26

44

34

3

28

24

43

33

4

28

25

43

36

4

28

25

43

36

Duodenal juice

 

 

 

 

 

0.4 mL

4

9

15

72

26

0.5 mL

4

14

20

62

40

*Glyceryl trioctanoate-1-14C was added to 1 mL of emulsified trioctanoin (60 μmoles) and incubated for 1 hour at 37ºC with buffer (blank) or gastric or duodenal juice. The reaction mixture was extracted and a 50 μL aliquot was analyzed by TLC and zonal scanning. A 3 mL aliquot was titrated to quantify fatty acids liberated.

Discussion:

The work confirmed extensive literature showing that gastric juice contains lipolytic activity, that ingested triglyceride is hydrolyzed in the stomach, even after pancreatic diversion, that lipase may be demonstrated histochemically in gastric mucosa, and that gastric mucosal homogenates have lipolytic activity. Pancreatic lipase has some activity at the pH of gastric content, which is between pH6 and pH3 in normal subjects.

Endpoint:
basic toxicokinetics
Type of information:
other: literature review
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary data Short review on metabolism from previous publications.
Objective of study:
metabolism

The metabolism of Medium chain triglycerides in the canine is a process whereby lipases from the buccal cavity and pancreas release the fatty acids in the gastrointestinal tract where they are absorbed. Unlike long chain triglycerides (LCT), where long chain fatty acids (LCFA) form micelles and are absorbed via the thoracic lymph duct, MCFA are most often transported directly to the liver through the portal vein and do not necessarily form micelles. Also, MCFA do not re-esterify into MCT across the intestinal mucosa. MCFA are transported into the hepatocytes through a carnitine-independent mechanism, and are metabolized into carbon dioxide, acetate, and ketones through b-oxidation and the citric acid cycle.

Endpoint:
basic toxicokinetics
Type of information:
other: review article
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: only secondary data
Reason / purpose:
reference to same study

Lipids are not only structural building blocks of cells and tissues but at the same time suppliers of C atoms for a number of biosynthetic pathways as well as carriers of essential fatty acids and fat-soluble vitamins. In addition, fatty acids are precursors of prostaglandins and other eicosanoids and therefore have important metabolic functions.

Fatty acids can be divided into three groups, saturated, monounsaturated, and polyunsaturated fatty acids.

Each class of fatty acids has a preferential specific role.

- Saturated fatty acids (medium or long-chain) are more devoted to energy supply, but one should not forget their specific structural role.

- The polyunsaturated fatty acids of the n–3 and n–6 families have very important structural and functional roles and ideally should not be utilized for energy purposes.

 

Table 1:

Role of different classes of fatty acids

Fatty acids

Energy

Structure

Function

Medium-chain saturated fatty acids

+++

0

0

Long-chain fatty acids

 

 

 

Saturated

++

++

(+)

Monounsaturated

++

++

(+)

Polyunsaturated

 

 

 

Linoleic or n-6 family

0

+++

+++

Linolenic or n-3 family

0

+++

+++

 0, +, ++, +++ : Emphasis of contribution, increasing in rank order

Description of key information

The toxicokinetic profile has been predicted using the physico-chemical properties of an analogue substance (EC 613 -848 -7), the data obtained from acute and repeated-dose toxicity studies, as well as information gained from genotoxicity assays. Further read-across to the toxicokinetic properties of fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is also applicable based on the similarity in structure and physico-chemical properties.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Introduction

The toxicokinetic profile of the analogue test item was predicted using the physico-chemical properties of an analogue substance (EC 613 -848 -7), the data obtained from acute and repeated-dose toxicity studies, as well as information gained from genotoxicity assays. Further read-across to the toxicokinetic properties of fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is also applicable based on the similarity in structure and physico-chemical properties.

 

Physico-chemical properties

 

The analogue test item is a UVCB, clear colorless liquid with the molecular weight of each of the four main isomers being 554.84 g/moL. The substance is poorly water soluble (2.04 x 10E-03 g/L) with a very high estimated octanol/water partition coefficient; Log Pow >9.4 (which is >4, the bioaccumulation limit), and a very low vapour pressure of 3.2 x 10E-06 Pa at 25 oC.

 

Absorption

 

Oral Route

 

The physico-chemical properties described above indicate that the analogue has a molecular size (MW >500) greater than that which may be expected to be easily absorbed within the mammalian gastrointestinal tract, should that material be ingested. The substance, which has a low water solubility and is highly lipophilic with a Log Pow>9.4) may be expected not to cross gastrointestinal epithelial barriers, and the high MW may also significantly restrict absorption. It may participate in micellar transport into the hepatic portal system along with other lipophilic substances (e.g., dietary fats). However, an acute oral gavage toxicity study identified no evidence of toxicity (LD50 >2000 mg/kg bw) and repeat dose and reproduction screening toxicology studies using the oral route gave a NOAEL of 1000 mg/kg bw/day. There was evidence of non-adverse, reversible effects in the liver and kidney, which supports a conclusion that some absorption does occur via the gastrointestinal tract. The lack of adverse findings following oral dosing may be due to limited gastrointestinal absorption of the test material after oral dosing, or a very low index of inherent toxicity for this substance, and/or its metabolite(s).

 

For the additional read-across substances, the absorbability of esterified alcohols containing one to eight ester groups given orally have been studied and there was little difference for polyol esters up to four ester groups. Esters of polyols (pentaerythritol, dipentaerythritol and 1,1,1-trimethylolpropane) have a common metabolic fate that involves stepwise hydrolysis to the carboxylic (e.g. fatty) acids and their polyols (pentaerythritol, dipentaerythritol or trimethylolpropane), respectively. This is supported by the action of ubiquitously distributed unspecific esterases and by site-specific and non-specific gastrointestinal lipases so only low and transient exposure to the parent compound is expected.

 

Dermal Route

 

Regarding the dermal absorption of the analogue test item, its rate of uptake into the stratum corneum and its rate of transfer between the stratum corneum and the epidermis are likely to be very slow considering both the high MW, high Log Powand low water solubility [1,2]. These assumptions were supported by the absence of observed systemic effects following dermal application of test material in an acute toxicity study at up to 2000 mg/kg bw and the absence of irritant effects to the skin.

 

Inhalation Route

 

The potential for inhalation toxicity was not studied directly in a toxicology study using the inhalation route. The analogue test substance has been shown to have a low vapour pressure and high onset boiling point range (did not boil up to 400 °C at 101 kPa). As a result, the potential for generation of inhalable forms of the substance is low and exposure of humans via the respiratory route is predicted to be negligible under normal use conditions. Furthermore, the Log Pow value of >9.4 does not favour absorption directly across the respiratory tract epithelium by passive diffusion (Log10 Pow > 4) and the substance will not be readily soluble in blood because it is poorly water soluble (≤ 2.04 x 10-3g/L at 20 °C).

 

Distribution

 

Systemic distribution of the analogue test item can be predicted from physico-chemical properties. The high Log Powand low water solubility suggests that this substance, upon systemic absorption, may be transported through the circulatory system in association with a carrier molecule such as a lipoprotein or other macromolecule. The lipophilic character but high Log Powand MW >500 of the test material suggests that a major proportion of the substance will not readily traverse cellular barriers or distribute into fatty tissues. There is no evidence of systemic toxicity and/or adverse histopathological changes from the repeated dose study that may be taken as evidence of cumulative toxicity, as would be expected by an accumulation of test item, or its metabolites, in body tissues.

 

Metabolism

 

Acute and repeated-dose toxicity studies provided evidence that the analogue test item was metabolised into non-toxic metabolites. Data from a bacterial mutagenicity test, a mammalian cell gene mutation assay and a chromosomal aberration in mammalian cells, in which test material was subjected to rat hepatic microsomal enzyme systems, did not show any evidence of genotoxic activity from the substance or from any potential metabolites. Furthermore, the in vitro toxicity of the substance to mammalian cells was equivalent in the presence and absence of metabolic enzymes. This may be taken to indicate either the absence of any significant level of metabolism, or that the metabolism that did occur gave rise to metabolites that were non-toxic.

 

Read-across to the toxicokinetic properties of fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. Esters of polyols (pentaerythritol, dipentaerythritol and 1,1,1-trimethylolpropane) have a common metabolic fate that involves stepwise hydrolysis to the carboxylic (e.g. fatty) acids and their polyols (pentaerythritol, dipentaerythritol or trimethylolpropane), respectively. This is supported by the action of ubiquitously distributed unspecific esterases and by site-specific and non-specific gastrointestinal lipases so only low and transient exposure to the parent compound is expected. Straight-chain fatty acids are normal dietary constituents and ubiquitous substrates for energy production by physiological pathways like the citric acid cycle, sugar synthesis, and lipid synthesis. Medium-chain fatty acids (MCFA) are readily absorbed from the small intestine directly into the bloodstream and transported to the liver for hepatic metabolism, while long-chain fatty acids (LCFA) are less readily absorbed, are incorporated into chylomicrons and enter the lymphatic system. It has been noted by several investigators that increasing fatty acid chain length slightly decreased their digestibility. MCFA are readily broken down to carbon dioxide and two-carbon fragments, while LCFA are re-esterified to triacylglycerols and either metabolized for energy or stored in adipose tissue. The natural occurrence of fatty acids and their specific metabolic fate imply that the exposure to small amounts is not a risk factor for human beings.

 

Excretion

 

Structural characteristics of the analogue test item and the toxicological profile suggest that this substance may readily undergo phase I and phase II metabolic transformation. The resulting metabolic by-products are expected to undergo routine renal and or biliary excretion. Metabolites such as polyols are very polar and do not accumulate in the body but are readily excreted via urine. Alternatively one or more hydroxyl groups can be oxidised to a carboxylic acid moiety prior to urinary excretion.

 

References

 

[1] Derivation of assessment factors for human health risk assessment. ECETOC Technical Repot No. 86. ISBN-0773-6347-86, Brussels, February 2003, page 13, paragraph 1.

 

[2] Guidance document on dermal absorption. European Commission Sanco/222/2000 rev. 7. Page 7, paragraph 2.