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Key value for chemical safety assessment

Effects on fertility

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Toxicity to reproduction is not expected.

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screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
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Justification for type of information:
The physiological characteristics of L-4-hydroxyproline (HYP) is its occurrence in collagen. Its content in collagen is high (ca. 12.4 %). Therefore, quantitative determination of HYP is even used for the verification of connective tissue material in meat products (Belitz et al., 2007).
HYP also occurs in plants as a component of such glycoproteins that are incorporated in cell walls (Belitz et al., 2007).

Besides in these main sources HYP also occurs in some animal proteins, e.g. acetyl cholinesterase (Anglister, et al., 1976), protein-bound HYP is present in e.g. bacteria (Chlamydomonas (Miller, et al., 1972).
As an amino acid made by post-translational hydroxylation of appropriate L-Proline residues, formation of the bulk of HYP in animals depends on the synthesis and maturation of collagen. This relationship influences every aspect of HYP metabolism. HYP is produced from L-Proline by pyrolyl hydroxylation with prolyl hydroxylase as the key enzyme (Cardinale and Udenfriend, 1974).
Ingested dietary protein – collagen in this case - 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 HYP is absorbed from ileum and distal jejunum.
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-proline 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 HYP concentrations in normal subjects are reported to be ca. 13 µM/L +/- 10 µM/L with plasma samples collected from healthy volunteers after an overnight fast (Cynober 2002).
Values of 0.04 – 0.13 µM/g are reported of rat liver (Haecock and Adams, 1975). Administered L-Proline has a depressing effect on the free HYP pool in liver (Haecock and Adams, 1975)
HYP released by gross collagen breakdown and subsequent hydrolysis of HYP-containing peptides cannot be directly reutilized for protein synthesis as it is committed to excretion or catabolism.
The major pathway of HYP catabolism in rat and beef tissue, and in man, was defined principally by enzymatic studies (Adams and Frank, 1980). Formation of 3-0H-P5C, the first oxidized product of HYP is catalyzed by mitochondria obtained from a number of mammalian species and. The pyrroline product can be reduced to HYP; the metabolic meaning of this reaction is obscure since it regenerates HYP from 3-0H-P5C whose only known source is HYP. This reduction may represent an analogous activity of the same enzyme that reduces P5C to L-Proline.
3-0H P5C is oxidized further to 4-0H-Olu. The latter compound was first recognized as a plant product in the threo-L-configuration. 4-0H-Glu is a substrate for several enzymes that act on Glu. One is Glu-aspartate transaminase. Other enzymes that act on 4-0H-Glu are glutamine synthetase, glutaminase, Glu-dehydrogenase, and Glu-decarboxylase both of animal and bacterial origin.

A minor mammalian pathway results in pyrrole-2-carboxylic acid via 4-OH-P2C as an intermediate (Haecock and Adams, 1975; Haecock and Adams, 1973).

HYP is limited essentially to collagen in humans, and its biosynthesis and catabolism, in bulk terms, are therefore inseparably linked to these processes for collagen. With this marker relationship, it was of obvious interest to measure HYP excretion under a variety of conditions affecting collagen metabolism. Urine is the relevant route of excretion. Major conclusions from these studies can be summarized as follows (Adams and Frank, 1980):

In normal man and other mammals studied, most of the HYP excreted is in the form of small peptides, approximating di- and tripeptides. A very small fraction of the total, consistently less than 5%, is free HYP. An additional minor fraction, 5 to 10% of the total, represents nondialyzable peptides up to at least 10,000 molecular weight. That the free HYP excreted is itself only a minor portion of that released from collagen is demonstrated in subjects lacking Hyp-oxidase, who excrete several hundred mg of free HYP daily, or 100 to 200 times the normal urinary level. These findings are in harmony with independent observations, that free HYP is efficiently reabsorbed by the kidney and rapidly catabolized via the major mammalian pathway; in contrast, peptide-HYP is efficiently excreted.

Individual peptides show varying resistance to further proteolytic cleavage, accounting in part for the particular pattern of peptides excreted in a given species. In summary, HYP releases as a free compound from protease-susceptible peptides is almost entirely catabolized; peptides resistant to proteolysis are largely excreted.

In man, total HYP excreted falls in a range of 15-50 mg/24 h for adults, with a sharp spike, up to 200 mg/24 h, in the adolescent years between age 10 to 20. Sex differences depend on body size and vanish by expressing results on a surface-area basis; the validity of referring HYP values to creatinine excretion has been questioned. While dietary collagen (especially gelatin-rich foods) can increase the daily HYP excretion, this increment does not generally exceed 20-30% over a HYP-free diet. Based on the excretion of free HYP in hyperhydroxyprolinemia, the rate of collagen turnover has been estimated as about 2 g/day, computed from the excretion of about 300 mg of HYP per day and the weight ratio of about 7 for collagen/HYP.

For risk assessment purposes oral absorption of HYP is set at 100%.

Other routes of excretion than that following to dietary intake are not relevant (e.g. exhalation).

HYP is of low volatility due to a very low vapour pressure (0.00000000152 mm Hg). From this and from the particle size it is not expected that HYP 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 131.13, any HYP reaching the lungs might be absorbed through aqueous pores (ECHA, 2014). For risk assessment purposes, although it is unlikely that HYP will be available to a high extent after inhalation via the lungs due to the low vapour pressure and high MMAD, the inhalation absorption of HYP is set at 100%.

Setting the dermal absorption rate of 10 % considers derivations in ECHA (2014). Initially, basic physico-chemical information should be considered, 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.

E. Adams and L. Frank (1980): Metabolism of Proline and the Hydroxyprolines (19809: Ann. Rev. Biochem. 49, 1005 - 1061

L. Anglister, S. Rogozinski, I. Silman (1976): FEBS letters 69, 699 - 704

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

G. Cardinale and S. Udenfriend (1974): Adv. Enzymol. 41, 245 - 300

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

Justification for classification or non-classification

Toxicity to reproduction is not expected.

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