1. health and fitness
2. disease

The EFSA Journal (2007) 473, 1-19
Opinion of the Scientific Panel on Additives and Products or
Substances used in Animal Feed on the safety and efficacy of the
product containing L-arginine produced by fermentation from
Corynebacterium glutamicum (ATCC-13870) for all animal species
(Question No EFSA-Q-2006-031)
Adopted on 17 April 2007
SUMMARY
The European Food Safety Authority (EFSA) received a request from the European
Commission to issue an opinion on the safety and the efficacy of a product containing Larginine produced by fermentation from Corynebacterium glutamicum (ATCC-13870)
modified by conventional methods. It contains a minimum of 98 % L-arginine on dry matter
basis (maximum 18% water).
L-arginine is classified as a non-essential amino acid for adult humans and most
mammalian species, but essential for mammalian neonates, strict carnivores, birds, fish
and possibly reptiles. Apart from its role as a component of protein synthesis, arginine has
many different functions in the metabolism.
The product can be used as a substitute of arginine from dietary protein. Demonstration of
efficacy of the product is not considered necessary by the FEEDAP Panel, because Larginine is a recognized nutrient. Taking into account published literature, the FEEDAP
Panel considers L-arginine from the product as bioavailable. The FEEDAP Panel concludes
that L-arginine gives no animal safety concern when used as a nutritional additive for all
animal species.
On the basis of the data available and considering the high degree of purity of the product
containing L-arginine, the FEEDAP Panel does not expect any risk for the consumer. The
product L-arginine was not mutagenic and showed no adverse effects up to 5% in the diet
of rats for 90 days. Increased deposition of L-arginine in animal products from the use of
the product is not expected.
The product contains no particles of less than 10 μm, thus potential for inhalation is not
considered of concern. No other data on user safety is provided but the manufacturer
considers the product may possess irritant properties and therefore proposes the use of
barrier protection methods.
L-arginine from the product is not expected to pose a risk to the environment.
The FEEDAP Panel does not see the need for additional requirements for a post-market
monitoring plan.
The FEEDAP Panel recommends refinements of the Register entry: (i) the additive should
be described as L-arginine, (ii) the description of the additive should be supplemented by ‘Larginine produced by fermentation from Corynebacterium glutamicum ATCC 13870’, (iii)
the minimum L-arginine content should be given on a dry matter basis (e.g. 98 % DM).
© European Food Safety Authority, 2007
Opinion on L-arginine for all animal species
2/19
The FEEDAP Panel also recommends the use of HPLC with post column derivatisation with
ninhydrin and photometric detection at 570nm for the determination of L-arginine in
feedingstuffs, and VDLUFA method 4.11.6 for the determination of the active substance in
the feed additive.
Key words:
L-arginine, Corynebacterium glutamicum, bioavailability, safety, nutritional
additive, amino acid
Opinion on L-arginine for all animal species
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TABLE OF CONTENTS
Summary .......................................................................................................................................................... 1
Table of Contents ............................................................................................................................................ 3
Background ...................................................................................................................................................... 4
Terms of reference.......................................................................................................................................... 4
Assessment...................................................................................................................................................... 6
1.
Introduction ............................................................................................................................................ 6
2.
L-arginine ................................................................................................................................................ 6
2.1.
The product V-Arginine ................................................................................................................... 6
2.2.
Physico-chemical properties ......................................................................................................... 7
2.3.
Analytical Methods ......................................................................................................................... 7
2.4.
Physiological significance and metabolism ............................................................................... 7
3.
Efficacy.................................................................................................................................................... 8
3.1.
Conclusion ........................................................................................................................................ 8
4.
Safety assessment ................................................................................................................................ 8
4.1.
Safety for the target animal .......................................................................................................... 8
4.2.
Safety studies on laboratory animals .......................................................................................... 9
4.2.1. Genotoxicity including Mutagenicity ....................................................................................... 9
4.2.2. Subchronic oral toxicity ............................................................................................................. 9
Safety for the consumer .............................................................................................................. 10
4.3.
4.4.
Safety for the user ........................................................................................................................ 10
4.5.
Safety for the environment.......................................................................................................... 10
5.
Post- Market Monitoring ..................................................................................................................... 10
Conclusions and Recommendations.......................................................................................................... 10
Documentation provided to EFSA ............................................................................................................... 11
References ..................................................................................................................................................... 12
Scientific Panel members............................................................................................................................ 13
Acknowledgements ...................................................................................................................................... 13
Appendices..................................................................................................................................................... 14
Opinion on L-arginine for all animal species
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BACKGROUND
Regulation (EC) No 1831/20031 establishes the rules governing the Community
authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that
Regulation lies down that any person seeking an authorisation for a feed additive or for a
new use of a feed additive shall submit an application in accordance with Article 7
The request for authorisation of L- Arginine has been submitted by the company Kyowa
Hakko Kogyo Europe GmbH.2 This application has been received on a first step according to
Directive 82/471/EEC. However, since October 2004, the amino acids, their salts and
analogues are covered by Regulation (EC) 1831/2003. Therefore, this application has been
transferred to the new regulatory provisions with a new request dated 31 March 2006. The
company Kyowa Hakko Kogyo Europe GmbH. is seeking authorisation of the product Larginine produced by fermentation of an isolated strain of Corynebacterium glutamicum
(ATCC-13870) and further isolation from the broth, de-colourisation, concentration,
crystallisation and drying (98% purity). The European Commission asks the product to be
added under the category ‘Nutritional’ and functional group ’Amino acids, their salts and
analogues’ for all species under the conditions described in Table 1.
According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the
application to the European Food Safety Authority (EFSA) as an application under Article
4.1 (authorisation of a feed additive or new use of a feed additive). EFSA received directly
from the applicant the technical dossier in support of this application. According to Article 8
of that Regulation, EFSA, after verifying the particulars and documents submitted by the
applicant, shall undertake an assessment in order to determine whether the feed additive
complies with the conditions laid down in Article 5. The particulars and documents in
support of the application were considered valid by EFSA as of 31 October 2006.
TERMS OF REFERENCE
According to Article 8 of Regulation (EC) No 1831/2003 EFSA, shall determine whether the
feed additive complies with the conditions laid down in Article 5. Therefore, EFSA shall
deliver an opinion on the efficacy and the safety for the target animals, the consumer, the
user and the environment of the product L-arginine, which is a preparation of L-arginine
produced by fermentation from Corynebacterium glutamicum (ATCC-13870) when used
under the conditions described in Table 1.
1
2
OJ L 268, 18.10.2003, p.29.
Kyowa Hakko Kogyo Europe GmbH, Immermannstrasse,3 D-40210, Dusseldorf, Germany
Opinion on L-arginine for all animal species
Table 1.
5/19
Register entry as proposed by the applicant
Additive
Arginine
Registration
number/EC
No/No (if appropriate)
-
Category of additive
Nutritional
Functional group of additive
Amino acids, their salts and analogues
Description
Chemical
formula
Composition, description
L-arginine, technically pure, 98%
Trade name
C6H14N4O2
Purity criteria
(if appropriate)
L-arginine minimum
80%
Method of analysis
(if appropriate)
Perchloric acid titration
V-Arginine
Name of the
authorisation
holder
of
Kyowa Hakko Kogyo Co, Ltd.
Conditions of use
Species or
category of
animal
Maximum
Age
All species
Minimum content
Maximum content
mg (or Units of activity or CFU) kg-1 of complete
feedingstuffs
-
-
Withdrawal
period
(if appropriate)
-
-
Other provisions and additional requirements for the labelling
Specific conditions or restrictions
for use (if appropriate)
Declaration to be made on the label or packaging of the
product: the name ‘L-arginine’
L-arginine’ content
Water content
Specific conditions or restrictions
for handling (if appropriate)
-
Post market monitoring
(if appropriate)
Specific conditions for use
complementary feedingstuffs
(if appropriate)
in
-
Maximum Residue Limit (MRL) (if appropriate)
Marker residue
Species or category
of animal
Target tissue(s) or
food products
Maximum content in
tissues
-
-
-
-
Opinion on L-arginine for all animal species
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ASSESSMENT
1.
Introduction
The applicant has requested authorisation for the product L-arginine for all species. Larginine is considered as a non-essential amino acid for adult humans and most adult
mammalian species, but it is classified as essential for birds, fish, possibly reptiles and also
for strict carnivores. For mammalian neonates, it is also considered to be essential.
The requirement under basal metabolism is between 1.0 and 1.6 % of essential arginine
for fast growing avian species and kittens, and between 0.1 and 0.6 % of the diet when
arginine is semi essential.
2.
L-arginine
The chemical formula of arginine is C6H14N4O2, the molecular weight 174.2.
Figure 1.
Chemical structure of arginine
L-arginine of microbial origin is an accepted and common supplement of human food and
animal feed in countries like the USA and Japan. The EU Scientific Committee for Food
found acceptable the use of L-arginine as a food for particular nutritional purposes (SCF,
1999).
2.1.
The product V-Arginine
The product described by the applicant as V-Arginine (L-arginine) is obtained from a
fermentation process with the strain Corynebacterium glutamicum (ATCC-13870). The
production strain was obtained by mutagenesis and was not subject to genetic
modification. Genetic manipulation such as recombinant DNA technology was not used.
The fermentation medium is a mixture of glucose syrup, ammonium sulphate, corn steep
liquor, phosphoric acid, and magnesium sulphate. No viable cells can be detected in the
broth after acidification with sulphuric acid. The product is subsequently purified using a
resin method, concentrated, crystallized and dried. Purity is given by the applicant with 98
% of the dry matter.
L-arginine is a white crystalline powder. The average particle size was 316 μm and there
were no particles of less than 50 μm. Dusting potential measured by the Stauber-Heubach
method is given with 9.7 mg per 25 gram of product indicating a very low dusting potential.
The L-arginine content of three batches of the product was assayed using a perchloric acid
titration technique and was shown to contain between 97.6 and 98.4 % of L-arginine on dry
matter basis. The same batches were also analysed for 17 free amino acids. The product
contained 0.02 - 0.04 % of free lysine, less than 0.01 - 0.02 % of free proline and less than
Opinion on L-arginine for all animal species
7/19
0.01 % for all other amino acids on a dry matter basis.3 Ammonium content was less than
0.019 %, determined by titration.4 More recent data (three batches, HPLC) did not show
detectable traces of lysine.5
The commercial product V-Arginine contains a minimum of 80 % L-arginine and not more
than 18 % water.
2.2.
Physico-chemical properties
A study with three lots of <18% moisture L-arginine under accelerated conditions (40°C
and 75% relative humidity)6 showed no losses after three months. The results indicate a
very high stability under extreme conditions.
The stability of L-arginine during processing and storage for three and six months in piglet
and sow feeds and premixes was also investigated.7 L-arginine is stable in feeds and
premixes during conventional processing of the feed and during at least 12 months of
storage under warehouse conditions.
2.3. Analytical Methods
EFSA has verified the Community Reference Laboratory (CRL) report on the Method(s) of
Analysis for L-arginine. The Executive Summary of the CRL report is attached as Appendix I.
2.4.
Physiological significance and metabolism
Despite its role as a component of protein synthesis, the importance of the dibasic amino
acid L-arginine in the intermediary metabolism as the precursor of ornithine and urea for
the detoxification of ammonia has been recognized since the discovery of the urea
(ornithine) cycle in 1932 (Krebs and Henseleit, 1932). In the past decades, additional vital
roles of L-arginine in physiology, nutrition and metabolic pathways as: the precursor of
proline, glutamate, creatine, nitric oxide (NO) and polyamines (putrescine, spermidine,
spermine), which are crucial to cell proliferation and differentiation; an allosteric activator
of N-acetylglutamate synthase (an essential cofactor for carbamoylphosphate synthase I),
and; a stimulator of the secretion of several hormones (e.g. insulin, growth hormone,
glucagon, prolactin), have been elucidated (see reviews by Wu and Morris, 1998; Wu et al.,
2000; Wu and Meininger, 2000).
On the basis of growth and N-balance, L-arginine is classified as a dispensable (nonessential) amino acid for adult humans and most adult mammalian species, but
indispensable (essential) for birds, fish and possibly for reptiles (Baby and Reddy, 1980)
because of the absence of an urea cycle, and also for strict carnivores because of the
absence of enzymes involved in the arginine synthesis (e.g. ornithine
carbamoyltransferase). Since the milk of almost all mammalian species examined to date
(cow, goat, sheep, elephant, llama, pig, rat), including primates (e.g. humans, gorillas,
chimps), is characterized by an arginine deficiency (Davis et al. 1994a,b; Wu and Knabe,
1994), dietary arginine supplementation can improve growth of milk-fed neonates, as
shown on piglets by Kim et al. (2004).
In view of the multitude of metabolites of physiological significance, which are synthesized
from arginine, N-balance and growth studies are not sufficiently sensitive to fully evaluate
3
4
5
6
7
Technical Dossier/Appendix E
Technical Dossier/Appendix E
Technical Dossier/July 2006/Appendix.9
Technical Dossier/July 2006/Appendix.6
Technical Dossier/July 2006/Appendix 7
Opinion on L-arginine for all animal species
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dietary arginine requirements (Wu and Morris, 1998). More details on the physiological
significance of L-arginine are shown in Appendix II.
3.
Efficacy
In the case of recognised nutrients/micro-nutrients, the FEEDAP Panel considers that no
demonstration of efficacy is needed, but in general evidence of bioavailability is required
No data on bioavailability was provided. The applicant submitted an expert review of one of
the leading arginine experts8 based on selected published articles. The expert is of the
opinion that L-arginine base has the same biological activity or bio-equivalency in animal
nutrition as other sources of L-arginine including L-arginine-HCl and peptide-bound Larginine in natural food proteins. The FEEDAP Panel supports this view, confirmed in
particular by the findings of Schuhmacher (2002).
The company submitted three field studies concerning the effect of arginine
supplementation of feeding sows on fertility. These studies do not provide any additional
contribution to the assessment of arginine as a nutritional additive.
3.1.
Conclusion
The FEEDAP Panel does not require further demonstration of efficacy because of the
character of L-arginine as a recognized nutrient. The FEEDAP Panel has no concern on the
bioavailability of L-arginine from the product (V-Arginine).
4.
Safety assessment
Toxicity studies with essential amino acids like arginine cannot be designed along the lines
of conventional toxicity experiments since the appearance of amino acid imbalances at
higher dosages will dramatically restrict the desired margin of safety.
4.1.
Safety for the target animal
The tolerance of animals to overdoses of amino acids varies with the amino acid
(substance and isomeric form) and the animal species. Excess of arginine (compared to the
classical requirement) is better tolerated than other amino acids, e.g. methionine. However,
arginine in excess (4 %) in pigs is less tolerated than equal excesses of lysine or threonine
(Edmonds et al., 1987). In young growing pigs, 1.63 % dietary arginine had no effect on
growth and feed intake and only affected feed efficiency, when lysine was marginally
deficient (1.03 % Lys; Hagemeier et al., 1983). In other studies with piglets, 0.67, 1.6 and
2.0 % supplemental arginine decreased weight gain and feed intake, but had variable
effects on gain/feed and no effect on the N-balance (Southern and Baker, 1982; Anderson
et al., 1984), whereas a moderate arginine supplementation (0.22 %) did not affect
performance of growing piglets (Rosell and Zimmerman, 1985). From experiment I on sows
(3 % supplemental arginine from day 14 until day 31 of pregnancy and from day 105 until
farrowing), a high tolerance could be concluded as well as from the subchronic study in rats
(5 % arginine for 13 weeks) 9. According to Baker (2004) excess arginine, is less growth
depressing in chicks than in pigs.
In broiler chicks, large excesses of arginine (4 %) reduced weight gain by only 9 % when
compared to a depression of 92, 79, 53, 50, 47, 31, 29, 15, 4 and 0 % by Met, Phe, Trp,
His, Lys, Tyr, Thr, Ile, Val and Leu, respectively (Edmonds and Baker, 1987). Furthermore, in
8
9
Wu G., Technical Dossier/August 2006/Appendix 14
Technical Dossier/Appendix M
Opinion on L-arginine for all animal species
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the same experiment, an addition of 1 % arginine improved the performance of chicks
receiving diets supplemented with 4 % excess Phe or Tyr.
The FEEDAP Panel concludes that L-arginine gives no cause for concern when used as a
nutritional additive for all animal species.
4.2.
Safety studies on laboratory animals
4.2.1.
Genotoxicity including Mutagenicity
The potential mutagenicity of three lots of L-arginine was examined by treating three
strains of S. typhimurium TA 98 and TA100 and E.coli WP2, at doses from 500 to 5000 μg
plate-1 in the absence and presence of metabolic activation, but not following the OECD
Guideline 471.10 The substances tested did not increase the number of revertants,
irrespective of the metabolic activation system. The positive control substances [AF-2:2-(2furyl)-3-(5-nitro-2-furyl) acrylamide and 2AA: 2-amino anthracene] remarkably increased the
number of revertants in the sensitive strains. It can be concluded that L-arginine is not
mutagenic under the conditions of this test.
The second study, in compliance with both OECD Guideline 471 and GLP, was performed in
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli
WP2uvrA- at up to 5000 μg plate-1 L-arginine, both with and without microsomal enzyme
activation systems, in three separate tests.11 The third test was conducted to check some
inconsistent results in the first two. Absence of any reproducible effects between the three
tests provided adequate reassurance on the lack of genotoxicity of L-arginine in this study.
A Chromosomal Aberration Test12 with L-arginine was performed in compliance with both
OECD Guideline 473 and GLP in a chromosome aberration test using cultured human
lymphocytes both with and without microsomal enzyme activation systems up to the 10
mM limit dose of 1740 μg mL-1. The test material showed no evidence of increased
chromosome aberrations with or without activation and was therefore considered to be
non-clastogenic to human lymphocytes under the conditions of the test.
4.2.2.
Subchronic oral toxicity
Food-grade L-arginine (99.8% purity) was administered to Crj: CD (SD) SPF rats (12 or
18/sex/group) for 13 weeks in diets containing 0 (control group: basal food), 1.25, 2.5 or
5.0 % added L-arginine.13 The reversibility of the possible toxic changes was also examined
after a five-week withdrawal period (control group and 5.0 % arginine group;
six/sex/group). The added levels were analytically confirmed. The estimated mean arginine
intake during the administration period in the 1.25, 2.5 and 5.0 % groups was 752, 1552
and 3,131 mg kg-1 body weight day-1 in males, respectively, and 910, 1803 and 3,565 mg
kg-1 body weight day-1 in females, respectively.
No dose-related changes were observed in clinical signs, body weight, food consumption,
feed efficiency or water intake. Also, ophthalmologic examination, urinalysis, haematology,
routine blood biochemistry, necropsy (12 females and 12 males), and histopathology did
not reveal any dose-related changes. Therefore, the NOAEL of L-arginine is concluded from
this study to be estimated at 5.0 % in diet (or 3,131 mg kg-1 body weight day-1).
In a four-week study, performed with Sprague-Dawley SPF rats [Crj:CD (SD) IGS] at six
weeks of age (15/sex/group), two L-arginine products of the same manufacturer were
10
11
12
13
Technical Dossier/Appendix P
Technical Dossier/August 2006/Appendix 17
Technical Dossier/August 2006/Appendix 18
Technical Dossier/Appendix Q
Opinion on L-arginine for all animal species
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compared.14 Both products are pharmaceutical grade products, showing a purity of 99.7
and 100 %. Both L-arginine products were administered orally by gavage at a dose of 2,000
mg kg-1 body weight day-1. The reversibility of the possible toxicity was examined in some
animals (five/sex/group) with a two-week withdrawal period.
No dose-related changes were observed in clinical signs, body weight, food consumption,
ophthalmologic examination, haematology, organ weights and necropsy. The number of
animals showing pH 9 in urine and positive protein reaction, increased in the female and
male arginine groups. Slight hyperplasia of the squamous epithelium at the limiting ridge in
the stomach was observed in both sexes. The changes seen were probably due to the
chosen route and method of administration of arginine. Changes either disappeared or
tended to recover by the end of the two-week withdrawal period, indicating that all the
effects seen in this study were reversible.
4.3.
Safety for the consumer
Evidence of the safety of L-arginine comes from the lack of effects in the toxicological
studies provided by the applicant: i) two GLP-compliant studies showing the lack of
mutagenicity; ii) a sub-chronic toxicity study that indicated that the additive was devoid of
adverse effect in the rat for doses up to 5 % in diet.
On the basis of the data available, and considering the high degree of purity of L-arginine
obtained using a multistep physico-chemical purification process, the FEEDAP Panel does
not expect any risk for the consumer. Increased deposition in animal products from the use
of L-arginine is not to be expected because i) synthetic arginine will mainly substitute
natural protein bound arginine and ii) body protein composition will not be affected by
supplemental dietary arginine.
4.4.
Safety for the user
The product contains no particles of less than 10 μm, thus potential for inhalation is not
considered of concern. No other data on user safety is provided but the manufacturer
considers the product may possess irritant properties and therefore proposes the use of
barrier protection methods.
4.5.
Safety for the environment
L-arginine is a physiological amino acid and a natural component of animals and plants
whose use in animal nutrition would not lead to any localized increase in concentration in
the environment. The FEEDAP Panel concludes that the use of the product as a feed
additive does not represent a risk to the environment.
5.
Post- Market Monitoring
The applicant will follow the requirements of the Feed Hygiene Regulation (Regulation (EC)
No 183/2005)15as well as the Good Manufacture Practice, which includes a system and
procedures for internal reporting. This being the case, the FEEDAP Panel does not see the
need for specific requirements for a post-market monitoring plan.
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
14
15
Technical Dossier/Appendix S
OJ L 35, 8.2.2005, p 1
Opinion on L-arginine for all animal species
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The product is a source of synthetic L-arginine able to replace arginine from natural
sources.
The FEEDAP Panel does not require further demonstration of efficacy because of the
character of L-arginine as a recognized nutrient. Taking into account published literature,
the FEEDAP Panel considers L-arginine from the product as bioavailable.
The FEEDAP Panel concludes that L-arginine gives no animal safety concern when used as
a nutritional additive for all animal species.
On the basis of the data available, and considering the high degree of purity of the product
containing L-arginine, the FEEDAP Panel does not expect any risk for the consumer. The
product L-arginine was not mutagenic and showed no adverse effects up to 5% in diet in
rats for 90 days. Increased deposition of L-arginine in animal products following the use of
the product is not to be expected.
The product contains no particles of less than 10 μm, thus potential for inhalation is not
considered of concern. No other data on user safety is provided but the manufacturer
considers the product may possess irritant properties and therefore proposes the use of
barrier protection methods.
L-arginine from the product is not expected to pose a risk to the environment.
The applicant will follow the requirements of the Feed Hygiene Regulation [Regulation (EC)
No 183/2005] as well as the Good Manufacture Practice, which includes a system and
procedures for internal reporting. This being the case, the FEEDAP Panel does not see the
need for specific requirements for a post-market monitoring plan.
RECOMMENDATIONS
The FEEDAP Panel recommends that the additive should be described as L-arginine and not
arginine, as is currently the case in the Register entry.
The description of the additive should be supplemented by “L-arginine produced by
fermentation from Corynebacterium glutamicum ATCC 13870.”
The FEEDAP Panel recommends that the minimum L-arginine content should be given on a
dry matter basis (e.g. 98 % DM).
The FEEDAP Panel recommends for official control purposes the use of HPLC with post
column derivatisation with ninhydrin and photometric detection at 570 nm for the
determination of L-arginine in feedingstuffs, and the Association of German Agricultural
Analytical and Research Institutes (VDLUFA) method 4.11.6 for the determination of the
active substance in the feed additive.
DOCUMENTATION PROVIDED TO EFSA
1.
L-arginine. Technical Dossier supporting the request of authorization under the
provisions of Council Directive 82/471/EEC including comments from the
Netherlands, UK and Germany, for all animal species. April 2005. Submitted by
Kyowa Hakko Kogyo co Ltd
2.
L-arginine. Answer to complementary information request. January 2007. Submitted
by Kyowa Hakko Kogyo co Ltd
3.
Evaluation report of the Community Reference Laboratory feed additives
authorisation on the methods(s) of analysis for L-arginine for all animal species
4.
Comments from the Member States submitted through the EFSAnet
Opinion on L-arginine for all animal species
12/19
REFERENCES
Anderson, L.C., Lewis, A.J., Peo, E.R. Jr, and Crenshaw, J.D. 1984. Effects of excess arginine
with and without supplemental lysine on performance, plasma amino acid
concentrations and nitrogen balance of young swine. J. Anim. Sci. 58(2), 369-77.
Baker, DH. 2004. Animal models for human amino acid responses. J. Nutr. 134, 1646S 1650S.
Baby, T.G. and Reddy, S.R.R. 1980. Arginine metabolism in purinotelic animals. I.
Characterization of arginase and absence of other urea cycle enzymes in the lizard,
Calotes versicolor. J. Comp. Physiol. B. 140(3), 261 - 265.
Davis, T.A., Nguyen, H.V., Garcia-Bravo, R., Fiorotto, M.L., Jackson, E.M., Lewis, D.S., Lee,
D.R. and Reeds, P.J. 1994a. Amino acid composition of human milk is not unique. J.
Nutr. 124(7), 1126 - 1132.
Davis, T.A., Nguyen, H.V., Garcia-Bravo, R., Fiorotto, M.L., Jackson, E.M. and Reeds, P.J.
1994b. Amino acid composition of the milk of some mammalian species changes with
stage of lactation. Br. J. Nutr. 72(6), 845 - 853.
Edmonds, M.S. and Baker, D.H. 1987. Comparative effects of individual amino acid
excesses when added to a corn-soybean meal diet: effects on growth and dietary choice
in the chick. J. Anim. Sci. 65(3), 699 - 705.
Edmonds, M.S., Gonyou, H.W. and Baker, D.H. 1987. Effect of excess levels of methionine,
tryptophan, arginine, lysine or threonine on growth and dietary choice in the pig. J. Anim.
Sci. 65(1), 179 - 85.
Hagemeier, D.L., Libal, G.W. and Wahlstrom, R.C. 1983. Effects of excess arginine on swine
growth and plasma amino acid levels. J. Anim. Sci. 57(1), 99 - 105.
Kim, S.W., McPherson, R.L. and Wu, G. 2004. Dietary arginine supplementation enhances
the growth of milk-fed young pig. J. Nutr. 134, 625 - 630.
Krebs, H.A. and Henseleit, K. 1932. Untersuchungen über die Harnstoffbildung im
Tierkörper [Studies on urea formation in the animal organism]. Hoppe-Seyler’s Z.
Physiol. Chem. 210, 33 - 66.
Kwon, H., Wu, G., Bazer, F.W. and Spencer, T.E. 2003. Developmental changes in polyamine
levels and synthesis in the ovine conceptus. Biol. Reprod. 69, 1626 - 1634.
Li, H., Meininger, C.J., Hawker, jr J.R., Haynes, T.E., Kepka-Lenhart, D., Mistry, S.K., Morris, jr
S.M., and Wu, G. 2001. Regulatory role of arginase I and II in nitric oxide, polyamine, and
proline syntheses in endothelial cells. Am. J. Physiol. Endocrinol. Metab. 280, E75–E82.
Morris, J.G. and Rogers, Q.R. 1978a. Ammonia intoxication in the near-adult cat as a result
of a dietary deficiency of arginine. Science 199(4327), 431 - 432.
Morris, J.G. and Rogers, Q.R. 1978b. Arginine: an essential amino acid for the cat. J. Nutr.
108(12), 1944 - 1953.
Morris, J.G., Rogers, Q.R., Winterrowd, D.L. and Kamikawa, E.M. 1979. The utilization of
ornithine and citrulline by the growing kitten. J. Nutr. 109(4), 724 - 729.
Morris, J.G. 1985. Nutritional and metabolic responses to arginine deficiency in carnivores.
J. Nutr. 115(4), 524 - 531.
Rogers, Q.R. and Phrang, J.M. 1985. Deficiency of pyrroline-5-carboxylate synthase in the
intestinal mucosa of the cat. J. Nutr. 115(1), 146 - 150.
Rosell, V.L. and Zimmerman, D.R. 1985. Threonine requirement of pigs weighing 5 to 15 kg
and the effect of excess methionine in diets marginal in threonine. J. Anim. Sci. 60(2),
480-6.
Schuhmacher, A. 2002. Limitierende Aminosäuren im Futter für wachsende Schweine [in
German]; Limiting amino acids in diets for growing pigs. Postdoctoral thesis, Faculty of
Veterinary Medicine, University of Leipzig 307 pp.
Scientific Committee on Food (SCF). 1999. Opinion on substances for nutritional purposes
which have been proposed for use in the manufacture of foods for particular nutritional
purposes (‘parnuts’). http://ec.europa.eu/food/fs/sc/scf/out31_en.pdf
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Seidel, E.R. and Scemama, J.L. 1997. Gastrointestinal polyamines and regulation of
mucosal growth and function. Nutr. Biochem. 8, 104 - 111.
Southern, L.L. and Baker, D.H. 1982. Performance and concentration of amino acids in
plasma and urine of young pigs fed diets with excesses of either arginine or lysine. J.
Anim. Sci. 55(4), 857- 66.
Stewart, P.M., Batshaw, M., Valle, D. and Walser, M. 1981. Effects of arginine-free meals on
ureagenesis in cats. Am. J. Physiol. 241(4), E310 – E315.
Windmueller, H.G., Spaeth, A.E. 1974. Uptake and metabolism of plasma glutamine by the
small intestine. J. Biol. Chem. 249(16), 5070 - 5079.
Windmueller, H.G. and Spaeth, A.E. 1975. Intestinal metabolism of glutamine and
glutamate from the lumen as compared to glutamine from blood. Arch. Biochem.
Biophys. 171(2), 662 - 672.
Windmueller, H.G., Spaeth, A.E. 1980. Respiratory fuels and nitrogen metabolism in vivo in
small intestine of fed rats. Quantitative importance of glutamine, glutamate, and
aspartate. J. Biol. Chem. 255(1), 107 - 112.
Wu, G., and Knabe, D.A. 1994. Free and protein-bound amino acids in sow’s colostrum and
milk. J. Nutr. 124(3), 415 - 424.
Wu, G., Knabe, D.A., and Flynn, NE. 1994. Synthesis of citrulline from glutamine in pig
enterocytes. Biochem. J. 299(1), 115 - 121.
Wu, G. and Knabe, D.A. 1995. Arginine synthesis in enterocytes of neonatal pigs. Am. J.
Physiol. 269(3), R621 - R629.
Wu, G. and Morris, jr S.M. 1998. Arginine metabolism: nitric oxide and beyond. Biochem. J.
336, 1 - 17.
Wu, G, and Meininger, C.J. 2000. Arginine and cardiovascular function. J. Nutr. 130, 2626 2629.
Wu, G., and Meininger, C.J., Knabe, D.A., Blazer, F.W. and Rhoads, J.M. 2000. Arginine
nutrition in development, health and disease. Curr. Opin. Clin. Nutr. Metab. Care. 3, 59 66.
Wu, G., Blazer, F.W., Cudd, T.A., Meininger, C.J. and Spencer, T.E. 2004. Maternal nutrition
and fetal development. J. Nutr. 134(9), 2169 - 2172.
Wu, G., Blazer, F.W., Hu, J. Johnson, G.A. and Spencer, T.E. 2005. Polyamine synthesis from
proline in the developing porcine placenta. Biol. Reprod. 72(4), 842 - 850.
SCIENTIFIC PANEL MEMBERS
Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro
Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle,
Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen,
Alberto Mantovani, Miklos Mezes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von
Wright and Pieter Wester.
ACKNOWLEDGEMENTS
The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes
to thank Dr. Annette Schuhmacher for her contribution to this opinion
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APPENDICES
APPENDIX I
Executive Summary of the Evaluation Report of the Community Reference Laboratory
Feed Additives Authorisation on the Method(s) of Analysis for L-arginine produced by
fermentation with Corynebacterium glutamicum (ATCC-13870)
In the current application authorisation is sought for L-arginine under the category
'nutritional additives', functional group 'amino acids, their salts and analogues', according
to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically,
authorisation is sought to use L-arginine for supplementing feed for all animal species. The
product is a crystalline powder with a minimum content of 80 % L-arginine. The feed
additive is intended to be included into feedingstuffs at a final concentration up to 5-6 % of
total L-arginine, depending on the concentration of L-arginine already present in the feed
components. For the determination of the active substance (L-arginine) in the feed additive
the applicant proposes a titrimetric method using perchloric acid. Since the feed additive
contains L-arginine in its base form, the CRL considers the more appropriate titrimetric
method using hydrochloric acid as prescribed by the Pharmacopea Europea. The applicant
also provides a High Performance Liquid Chromatography (HPLC) method which is specific
for the analyte, but without specifying related validation data. For official control purposes,
the CRL recommends validated methods based on the same technique, such as the
method 4.11.6 of the Association of German Agricultural Analytical and Research Institutes
(VDLUFA) [Methodenbuch III, 5. Erg. 2004, VDLUFA – Verlag, Darmstadt ] and the similar
AOAC Method 999.13 [Fontaine and Eudaimon, J. of AOAC Int., Vol. 83, No. 4, 2000]. These
methods have been validated for the quantitative determination of three free (non protein
bound) amino acids (lysine, methionine and threonine) in feed grade amino acid
commercial products and premixtures with more than 10 % individual amino acid content,
and can be applied also for the determination of L-arginine.
The applicant does not describe whether the additive is intended to be directly incorporated
into feedingstuffs or through premixtures. However, for the determination of L-arginine in
premixtures for official controls, the same above mentioned Official or validated methods
are recommended by the CRL.
For the determination of the active substance (L-arginine) in feedingstuffs the applicant
proposes the official Community and fully ring-trial validated method for determination of
amino acids [Commission Directive 98/64/EC]. The method is applicable for both the
determination of free (synthetic and natural) and the determination of total (peptide-bound
and free) amino acids, using an amino acid analyser or HPLC equipment with post column
derivatisation with ninhydrin and photometric detection at 570 nm. The same method is
adopted by ISO and described in the ISO standard 13903:2005 [Animal feedingstuffs –
determination of amino acids content - ISO 13903:2005], which additionally reports the
results from a second intercomparison study performed on different premixtures and feeds
[Llames & Fontaine, J. of AOAC Int., Vol. 77, No. 6, 1994]. Performance characteristics for
the target analyte (L-arginine) include the relative repeatability standard deviation (RSDr)
ranging from 2.34 to 3.31 % and relative reproducibility standard deviation (RSDR) ranging
from 7.18 to 9.66 %, depending on the matrix. The method does not distinguish between
the salts of amino acids, nor differentiates between D and L forms of amino acids. The
method is considered suitable for official controls.
Further testing or validation by the CRL is not considered necessary.
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APPENDIX II
Function and metabolism of Arginine
A II. 1 Biosynthesis
Arginine can be synthesized from glutamine, glutamate, citrulline, ornithine and proline as
demonstrated in Figure II.1. However, the complete pathway shown in Figure A II.1 is only
present in the small intestine of neonates and in pigs after weaning. In other adult animals
and humans, the endogeneous de novo synthesis of arginine, which accounts for only 5 15 % of the endogeneous arginine flux in adults, involves an inter-organ pathway, the
intestinal-renal axis. Glutamine extracted from plasma, and glutamine and glutamate
derived from the intestinal lumen are extensively catabolized to citrulline or arginine in the
enterocytes of the small intestine (Windmueller and Spaeth, 1974, 1975, 1980; Wu et al.,
1994; Wu and Knabe, 1995). In contrast, citrulline synthesis and its release is impaired in
the small intestine of strictly carnivorous species, such as cats; the activities of the L-δ1pyrroline-5-carboxylate (P5C) synthase and ornithine aminotransferase (OAT) are relatively
low as compared to other mammals (Morris and Rogers, 1978a,b; Morris et al., 1979;
Stewart et al., 1981; Morris, 1985; Rogers and Phrang, 1985), which results in a dietary
arginine requirement of carnivores.
Another pathway of arginine synthesis, at least in pigs, is the conversion of proline to
arginine by the action of proline oxidase in the small intestine (Wu and Morris, 1998).
Circulating citrulline is extracted by the kidney for the synthesis of arginine, which accounts
for approximately 60 % of the net arginine synthesis in adult mammals; the renal capacity
for arginine synthesis increases continuously during the early stages of life accompanied by
a concomitant decrease of the intestinal arginine synthesis (Wu and Morris, 1998).
Conversely, the contribution of liver to the net arginine synthesis is small because of the
efficient utilization of the metabolites of the urea cycle, which are channelled from one
enzyme to the next.
Furthermore, in non-hepatic NO-producing cells, citrulline can be recycled to arginine via the
so-called citrulline/NO cycle or arginine/citrulline cycle (Wu and Morris, 1998); that
pathway of arginine synthesis is regulated by L-glutamine (competitive inhibitor of citrulline
uptake) and hypoxia (reduction of agininosuccinate synthase). The cycle as according to Wu
and Morris (1998) and by Li et al. (2001) is summarized in Figure II.2.
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Figure II.1.
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Pathways of arginine synthesis (Wu and Morris, 1998)
Enzymes that catalyse the indicated reactions are: 1, phosphate-dependent glutaminase; 2 and 3,
P5C synthetase; 5, ornithine aminotransferase (OAT); 6, ornithine carbamoyltransferase (OCT); 7,
argininosuccinate synthase (ASS); 8 argininosuccinate lyase (ASL); 9 N-acetylglutamate synthase;
10 carbamoyl-phosphate synthase I (ammonia) (CPS I); 11 proline oxidase; 12 aspartate
aminotransferase. Step 4 is a spontaneous, non-enzymic reaction. Glutamyl-c-semialdehyde is in
chemical equilibrium with P5C. The chemical equilibrium favours P5C formation. P5C synthetase is
a bifunctional polypeptide that exhibits both c-glutamyl kinase (glutamate 5-kinase) and cglutamylphosphate reductase (glutamate-5-semialdehyde dehydrogenase) activities (reactions 2
and 3 respectively). Reactions 1±6 and 9±11 occur in mitochondria, reactions 7 and 8 take place in
the cytosol, and reaction 12 can occur in both mitochondria and the cytosol. Abbreviations: OAA,
oxaloacetate; CP carbamoyl phosphate.
Opinion on L-arginine for all animal species
Figure II.2.
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Arginine metabolism in mammalian cells (Li et al., 2001; Wu and Morris,
1998)
ASL, argininosuccinate lyase; Asp, aspartate; ASS, argininosuccinate synthase; BH4,
tetrahydrobiopterin; DCAM, decarboxylated 5-adenosylmethionine; Glu, glutamate; MTA,
methylthioadenosine; NO, nitric oxide; NOS, nitric oxide synthase; OAT, ornithine aminotransferase;
P5CD, pyrroline-5-carboxylate dehydrogenase; P5CR, pyrroline-5-carboxylate reductase; α-KG, α ketoglutarate.
A II. 2 Catabolism and physiological effects
The catabolism of arginine via multiple pathways, which are possibly regulated by Type I
arginase (cytosol; mainly expressed in liver) and Type II arginase (mitochondria; at lower
extent in kidney, brain, small intestine, mammary gland, macrophages), results in a variety
of metabolic active substances as shown in Figure II.3 (and in Figure II.2); almost all of
these metabolites, obviously, may show beneficial effects on the development and health
of humans and animals. The role of agmatine in the mammalian organism is not fully
understood; it is possibly a weak regulator of NO and polyamine synthesis (Wu and Morris,
1998). Polyamines (putrescine, spermidine, spermine) are essential for cell proliferation,
differentiation and function (scavengers of reactive oxygen species), and are therefore key
regulators of angiogenisis, placental growth and embryonic development (Kwon et al.,
2003). Thus, impaired placental syntheses of polyamines and NO due to maternal
undernutrition and overnutrition may explain fetal growth retardation predisposing the
offspring to metabolic, endocrine and cardiovascular diseases induced by stable alteration
of gene expression (Wu et al., 2004). However, findings published recently indicate that
proline is the major amino acid for polyamine synthesis and not arginine, because in
porcine placenta neither an arginase activity nor a conversion of arginine to polyamines
could be detected (Wu et al., 2005). Polyamines may also be produced by gut flora and
may significantly contribute to the body polyamine pool (Seidel and Scemama, 1997).
Nitric oxide, an endothelium-derived relaxing factor, is co-synthesized with citrulline from
arginine by the action of one of the isoforms of NO synthase (NOS) which are present in
almost all mammalian cells (see Table II.1). NO is essential for the regulation of vascular
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tone (vasodilatation) and hemodynamics, and therefore for cardiovascular function;
arginine plays also important roles in wound healing (stimulation of the proliferation of
endothelium cells, angiogenisis), immune responses, neurotransmission and adhesion of
platelets and leukocytes (Wu and Morris, 1998; Wu and Meininger, 2000; Wu et al., 2000).
Figure II.3.
Metabolic fates of arginine in mammalian cells (Wu and Morris, 1998)
The five enzymes on which the central limbs of the pathways are based include (clockwise from the
top): nitric oxide synthase (NOS), arginine : glycine amidinotransferase, arginase, arginine
decarboxylase and arginyl-tRNA synthase.
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Further effects of the arginine metabolites are given in Table A II.1.
Table II.1 Nitric oxide (NO) dependent and independent vascular effects of L-arginine
according to Wu and Meininger (2000)
NO-dependent
Smooth muscle cell relaxation
EC proliferation and angiogenisis
Endothelin-1 relaxase
Leukocyte adhesion
Platelet aggregation
Superoxide production
Expression of cell adhesion molecules
Expression of monocyte chemotactic peptides
Proliferation of smooth muscle cells
EC apoptose
NO-independent
Polarization of EC membranes
Extracellular and intracellular pH
Release of Insulin, GH, glucagons and polyamines
Synthesis of urea, creatine, proline and polyamines
Plasmin generation and fibrinogenolysis
Leukocyte adhesion to non-EC matrix
Blood viscosity
Angiotensin-converting enzyme activity
O2 - release and lipid peroxidation
Formation of TXB2, fibrin and platelet-fibrin
EC, endothelial cells; GH, growth hormone; TXB2, thromoxane B2.