Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
other: Toxikokinetic assessment
Adequacy of study:
key study
Study period:
March 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Toxicokinetic assessment

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2012

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Toxicokinetic assessment, based on
- Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance
- ECB EU Technical Guidance Document on Risk Assessment, 2003
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
L-alanine
EC Number:
200-273-8
EC Name:
L-alanine
Cas Number:
56-41-7
Molecular formula:
C3H7NO2
IUPAC Name:
(2S)-2-aminopropanoic acid

Results and discussion

Main ADME results
Type:
absorption
Results:
Oral route: 100%. Dermal route: 10 %. Inhaltion route: 100 %

Any other information on results incl. tables

L-alanine is a natural occurring amino acid and a natural constituent of peptides and proteins. Its content in most proteins is ca. 2 – 7 %, in gelatine and remarkably high in silk fibroin (ca. 35 %) (Belitz et al, 2007). L-alanine belongs to the group of amino acids with uncharged non-polar side chains. It is non-essential in humans.

L-Alanine is most commonly produced by reductive amination of pyruvate. Because transamination reactions are readily reversible and pyruvate pervasive, alanine can be easily formed and thus has close links to metabolic pathways such as glycolysis, gluconeogenesis, and the citric acid cycle. It also arises together with lactate and generates glucose from protein via the alanine cycle. (Lehninger et al, 2000)

 

 

Adsorption

L-alanine is absorbed from the gastrointestinal tract. Ingested dietary protein is denatured in the stomach due to low pH. Denaturing and unfolding of the protein makes the chain susceptible to proteolysis. Up to 15% of dietary protein may be cleaved to peptides and amino acids by pepsins in the stomach. In the duodenum and small intestine digestion continues through hydrolytic enzymes (e.g. trypsin, chymotrypsins, elastase, carboxypeptidase). The resultant mixture of peptides and amino acids is then transported into the mucosal cells by specific carrier systems for amino acids and for di- and tripeptides.

 

The products of digestion are rapidly absorbed. Like other amino acids L-alanine is absorbed from ileum and distal jejeunum.

 

 

Distribution

Absorbed peptides are further hydrolysed resulting in free amino acids which are secreted into the portal blood by specific carrier systems in the mucosal cell. Alternatively they are metabolised within the cell itself. Absorbed amino acids pass into the liver where a portion of the amino acids are used. The remainder pass through into the systemic circulation and are utilised by the peripheral tissue. L-alanine is actively transported across the intestine from mucosa to serosal surface. The mechanism of absorption is that of the ion gradient. All L-amino acids are absorbed by Na+dependant, carrier mediated process. This transport is energy dependant by ATP. (All data from: Lehninger et al, 2000; Chatterjea and Shinde, 2012.)

 

Plasma L-alanine concentrations in normal subjects are reported to be ca. 333 µM/L +/- 74 mM/L with plasma samples collected from healthy volunteers after an overnight fast; Cynober 2002). As with most nutrients, plasma concentration of L-alanine is subject to homeostasis.

 

A number of hormones (e.g., thyroid hormone, catecholamines, and growth hormone) may affect plasma AA levels in diseases (Cynober et al., 1987). However, in the physiologic state, their influence is probably marginal. However, there is the counter-regulatory hormon system with cortisol and glucagon which influences the blood level of amino acids involved in gluconeogenesis, such as L-alanine (Boden et al., 1984).

 

 

Metabolism

There is no storage form for amino acids in animals and human except in the biologically active protein of the cells.

 

Alanine is most commonly produced by the reductive amination of pyruvate via alanine transaminase. This reversible reaction involves the interconversion of alanine and pyruvate, coupled to the interconversion of alpha-ketoglutarate (2-oxoglutarate) and glutamate. Because transamination reactions are readily reversible and pyruvate is widespread, alanine can be easily formed in most tissues. Another route to the production of alanine is through the enzyme called alanine-glyoxylate transaminase. This reaction involves the interconversion of alanine and pyruvate, coupled to the interconversion of glyoxylate and glycine. Once synthesized, alanine can be coupled to alanyl tRNA via alanyl-tRNA synthetase and used by the body in protein synthesis. Alanine constitutes about 8% of human proteins. Under fasting conditions, alanine, derived from protein breakdown, can be converted to pyruvate and used to synthesize glucose via gluconeogenesis in the liver. Alternately, alanine, after conversion to pyruvate, can be fully oxidized via the TCA cycle in other tissues (Lehninger, 2010; Salway, 2004)

 

 

Excretion

Body losses of amino acids are minimal because amino acids filtered by the kidneys are actively reabsorbed. Also cutaneous losses and losses via exhalation are negligible. Since there is no long term storage for amino acids in mammals, excess amino acids are degraded, mainly in the liver. Metabolism of amino acids involves removal of the amino group which is converted to urea and excreted in the urine. After removal of the amino group the rest of the acid is utilised as energy source or in anabolism of other endogenous substances.

 

Alanine plays a key role in glucose–alanine cycle between tissues and liver. In muscle and other tissues that degrade amino acids for fuel, amino groups are collected in the form of glutamate by transamination. Glutamate can then transfer its amino group through the action of alanine aminotransferase to pyruvate, a product of muscle glycolysis, forming alanine and α-ketoglutarate. The alanine formed is passed into the blood and transported to the liver. A reverse of the alanine aminotransferase reaction takes place in liver. Pyruvate regenerated forms glucose through gluconeogenesis, which returns to muscle through the circulation system. Glutamate in the liver enters mitochondria and degrades into ammonium ion through the action of glutamate dehydrogenase, which in turn participate in the urea cycle to form urea. The glucose–alanine cycle enables pyruvate and glutamate to be removed from the muscle and find their way to the liver. Deficiency in supply of L-alanine may cause health disorders (Nelson and Cox, 2005).

 

The concentration of L-alanine in blood after uptake is relatively constant (Franchi-Gazzola et al., 1982).

Intestinal absorption rates of amino acids were found to be > 50 % for most amino acids (Adibi et al, 1967) whereby the absorption rate of individual amino acids may increase depending of the administered concentration. These findings were made with amino acid concentrations far below the saturation plateau of the transport system. The absorption rate expressed as g/h of amino acids from different dietary proteins is high (Bilsborough and Mann, 2006) and indicates absorption rates > 50 % for individual amino acids. An oral absorption rate of 100 % is more realistic than a rate of 50 %.

The amounts of protein and, therefore, of amino acids consumed by humans vary over a wide range. When dietary nitrogen and essential amino acid intakes are above the requirement levels, healthy individuals appear to adapt well to highly variable dietary protein intakes, because frank signs or symptoms of amino acid excess are observed rarely, if at all, under usual dietary conditions (Bier, 2003). When considering the fact that DNELs based upon 100 % absorption rate are by factors well lower than the daily dietary intake of humans, an absorption rate of 100 % is not only realistic for the purpose of safety assessment but sufficiently safe.

For risk assessment purposes oral absorption of L-alanine is set at 100%.

 

L-alanine is of low volatility due to a very low vapour pressure (0.00000283 Pa). From this and from the particle size it is not expected that L-alanine reaches the nasopharyncheal region or subsequently the tracheobronchial or pulmonary region in significant amounts.

 

However, being a very hydrophilic substance with a molecular mass of only 89.09, any L-alanine reaching the lungs might be absorbed through aqueous pores (ECHA, 2008). For risk assessment purposes, although it is unlikely that L-alanine will be available to a high extent after inhalation via the lungs due to the low vapour pressure and high MMAD (Mass median aerodynamic diameter), the inhalation absorption of L-alanine is set at 100%.

 

L-alanine with high water solubility (166.5 g/L) and the log P value below 0 (- 2.74) may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Therefore, 10% dermal absorption of L-alanine is proposed for risk assessment purposes.

 

Setting the dermal absorption rate tof 10 % considers derivations in ECHA (2008). Initially, basic physico-chemical information should be taken into account, i.e. molecular mass and lipophilicity (log P). Following, a default value of 100% skin absorption is generally used unless molecular mass is above 500 and log P is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen (de Heer et al, 1999). The lower limit of 10% was chosen, because there is evidence in the literature that substances with molecular weight and/or log P values at these extremes can to a limited extent cross the skin.

For substances with log P values <0, poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low.

 

Citations:

Adibi S.A.,Gray S.J.,Menden E. (1967). The kinetics of amino acid absorption and alteration of plasma composition of free amino acids after intestinal perfusion of amino acid mixtures. Am J Clin Nutr. 1967 Jan; 20 (1): 24-33.

Belitz H.-D., Grosch W. und Schieberle P. (2007): Lehrbuch der Lebensmittelchemie. 6. Auflage. Springer-Verlag, Berlin und Heidelberg

Bier, D.M. (2003): Amino acid pharmacokinetics and safety assessment. J. Nutr. 133: 2034S-2039S.

Bilsborough, S. and Mann, N. (2006): A review of issues of dietary protein intake in humans. International Journal of Sport nutrition and Excercise Metabolism, 16, 129-152

Boden G., Rezvani I., Owen O.E. (1984): Effects of glucagon on plasma amino acids. J Clin Invest ,73:785

 

Chatterjea M. and Shinde R. (2012): Textbook of Medical Biochemistry. Jaypee Brothers Medical Publishers, New Delhi

 

Cynober L. (2002): Plasma Amino Acid Levels With a Note on Membrane Transport: Characteristics, Regulation, and Metabolic Significance. Nutrition 18 (9), 761-766

 

Cynober L., Coudray-Lucas C., Ziegler F., et al. (1987): Metabolisme azote´ chez le sujet sain. Nutr Clin Metabol;.3: 87

De Heer C., Wilschut A., Stevenson H., and Hakkert B. C. Guidance document on the estimation of dermal absorption according to a tiered approach: an update.V98. 1237. 1999. Zeist, NL, TNO. 

ECHA (2008): Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance

 

Renata Franchi-Gazzola F., Gazzola G., Dall’Asta V., and Guidottj G. (1982): The Transport of Alanine, Serine, and Cysteine in Cultured Human Fibroblasts. J. Biological Chemistry 257, No. 16, Issue of August 25. 9582-9587

 

Lehninger A., Nelson D., Cox M. (2000), Principles of Biochemistry (3rd ed.), New York: W. H. Freeman

 

Salway J.G. (2004): Metabolism at a glance (3rd ed.). Alden, Mass. : Blackwell Pub.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: The absorption factors for risk assessment purposes have been set as follows: absorption oral 100%, absorption dermal 10% and absorption inhalation 100%
For risk assessment purposes:
Absorption oral: 100%,
Absorption dermal: 10%
Absorption inhalation: 100%