SCIENTIFIC OPINION  
ADOPTED: 27 January 2016   PUBLISHED: 19 February 2016  
doi:10.2903/j.efsa.20 16.4394   
 
 
www.efsa.europa.eu/efsajournal   EFSA Journal 20 16;14(2):4394 
 
Safety and efficacy of guanidinoacetic acid for chickens 
for fattening, breeder hens and roosters, and pigs  
EFSA Panel on Additives and Products or Substances used in 
Animal Feed (FEEDAP)  
Abstract  
Guanidinoacetic acid (GAA) occurs naturally in the body of animals (and humans) and acts as a 
precursor of creatine. The safe level of the product appears to be correlated with the supply of methyl 
donors. If an adequate supply of methyl donors is provided in the feed, a safe level of 1 ,200 mg 
GAA/kg complete feed could be established for chickens for fattening and piglets. The conclusion on 
piglets can be extended to pigs for fattening. In the absence of adequate data, the FEEDAP Panel 
cannot conclude on the safety of GAA for breeder hens and roosters. GAA is not mutagenic. The 
effects reported in the 28 - and 90 -day studies do not identify any unexpected toxicity other than 
physiological effects. Feeding GAA would not result in GAA, creati ne or homocysteine concentrations 
in chicken and pig tissues/organs relevant to consumer safety provided the maximum GAA 
concentrations in feed considered safe are respected. GAA is not an irritant to eyes and skin and not a 
dermal sensitiser. Significant inhalation exposure may occur. However , due to the absence of data on 
respiratory toxicity , no risk assessment can be performed. The use of GAA in animal nutrition is not 
expected to pose a risk to the environment. Considering the data previously assessed and the two 
new studies, the FEEDAP Panel concludes that GAA is able to improve the performance in chickens for 
fattening at the minimum dose of 600 mg/kg. In the absence of adequate data, the FEEDAP Panel 
cannot conclude on the efficacy of GAA in breeder hens and roosters. In piglets, only one study was 
available showing that 1 ,200 mg GAA/kg complete feed improved growth. Therefore , the FEEDAP 
Panel is not in a position to conclude  on the efficacy of GAA in this species . 
© European Food Safety Authority, 2016 
 
Keywords:  nutritional additive, guanidinoacetic acid, CreAMINO, safety, efficacy, homocysteine, 
chicken, pig  
Requestor:  European Commission  
Question number:  EFSA-Q-2012-00273  
Correspondence:  feedap @efsa.europa.eu  
 
 
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Panel members:  Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, 
Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Jürgen Gropp, Boris 
Kolar, Maryline Kouba, Secundino López Puente, Marta López -Alonso, Alberto Mantovani, Baltasar 
Mayo, Fernando Ramo s, Guido Rychen, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace 
and Pieter Wester  
Acknowledgements:   The Panel wishes to thank the members of the Working Group on Amino 
Acids and Vitamins including Lucio Costa , Noël Dierick  and Lubomir Leng ; as well as the former 
members of the Working Group  on Amino Acids , Paul Brantom and Giovanna Martelli , for the 
preparatory work on this scientific opinion and EFSA staff  member  Jordi Tarrés -Call for the support 
provided to this scientific opinion.  
Amendment:  An editorial amendment was carried out to delete two contradictory sentences about 
the efficacy in pigs on p.21, immediately after Table 7. To avoid confusion, the older version has been 
removed from the EFSA Journal, but is available on request, as is a version sh owing all the changes 
made.  
Suggested citation:  EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used 
in Animal Feed ), 20 16. Scientific opinion on the safety and efficacy of guanidinoacetic acid for 
chickens for fattening, breeder hens  and roosters, and pigs . EFSA Journal 20 16;14(2):4394 , 39 pp. 
doi:10.2903/j.efsa.20 16.4394 
ISSN:  1831-4732 
© European Food Safety Authority, 2016 
Reproduction is authorised provided the source is acknowledged.  
 
 The EFSA Journal is a publication of the European Food  
 Safety Authority, an agency of the European Union.   
 
 
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Summary  
Following a request from the European Commission , the Panel on Additives and Products or 
Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and 
efficacy of guanidinoacetic acid (GAA; CreAMINO®) for chickens for  fattening, breeder hens and 
roosters, and pigs.  
CreAMINO®, a granulated product, contains a minimum of 96% chemically synthesised GAA as active 
substance. Glycine (≤ 1.5%), cyanamide ( ≤ 0.03%), dicyandiamide ( ≤ 0.5%) and melamine 
(≤ 0.002%) are impurities  arising from the production process.  
GAA, a glycine derivative, occurs naturally in the body of animals (and humans) and acts as a 
precursor of creatine. The metabolic step from GAA to creatine requires methyl groups (from methyl 
donors) and methyl -transf erring enzymes.  
The safe level of the product appears to be correlated with the supply of methyl donors. If an 
adequate supply of methyl donors is provided in the feed, a safe level of 1 ,200 mg GAA/kg complete 
feed could be established for chickens for fat tening and piglets. The conclusion on piglets can be 
extended to pigs for fattening. In the absence of adequate data, the FEEDAP Panel cannot conclude 
on the safety of GAA for breeder hens and roosters.  
GAA is not mutagenic. The effects reported in the 28 - and 90 -day studies do not identify any 
unexpected toxicity other than physiological effects. Feeding GAA would not result in GAA, creatine or 
homocysteine concentrations in chicken and pig tissues/organs relevant to consumer safety provided 
the maximum GA A concentrations in feed considered safe are respected.  
GAA is not an irritant to eyes and skin and not a dermal sensitiser. Significant inhalation exposure may 
occur. However , due to the absence of data on respiratory toxicity , no risk assessment can be 
performed.  
The use of GAA in animal nutrition is not expected to pose a risk to the environment.  
Considering the data previously assessed and the two new studies, the FEEDAP Panel concludes that 
GAA is able to improve the performance in chickens for fattening at the minimum dose of 600 mg/kg. 
In the absence of adequate data, the FEEDAP Panel cannot conclude on the efficacy of GAA in 
breeder hens and roosters. In piglets, only one study was available showing that 1 ,200 mg GAA/kg 
complete feed improved growth. When the assessment is based on zootechnical parameters, at least 
three studies are required ; therefore , the FEEDAP Panel is not in a position to conclude on the basis of 
this single study.  
The W orking Group made two recommendations (one on the spe cification of the additive , and the 
other on the need to ensure that the feed contains sufficient methyl donors other than methionine) . 
  
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Table of contents  
 
Abstract  ................................ ................................ ................................ ................................ .........  1 
Summary  ................................ ................................ ................................ ................................ ....... 3 
1. Introduction  ................................ ................................ ................................ ........................  5 
1.1. Background and Terms of Reference  ................................ ................................ ....................  5 
1.2. Additional information  ................................ ................................ ................................ .........  5 
2. Data and methodologies  ................................ ................................ ................................ ...... 5 
2.1. Data ................................ ................................ ................................ ................................ ... 5 
2.2. Methodologies  ................................ ................................ ................................ ....................  6 
3. Assessm ent ................................ ................................ ................................ ........................  6 
3.1. Characterisation  ................................ ................................ ................................ ..................  6 
3.1.1.  Manufacturing process  ................................ ................................ ................................ ........  6 
3.1.2.  Characterisation of the active substance  ................................ ................................ ...............  6 
3.1.3.  Characterisation of the formulated additive  ................................ ................................ ...........  7 
3.1.4.  Stability and homogeneity  ................................ ................................ ................................ ... 8 
3.1.5.  Physico -chemical incompatibilities in feed  ................................ ................................ .............  9 
3.1.6.  Conditions of use  ................................ ................................ ................................ ................  9 
3.2. Safety  ................................ ................................ ................................ ................................  9 
3.2.1.  Safety for the target species  ................................ ................................ ................................  9 
3.2.2.  Safety for the consumer  ................................ ................................ ................................ .... 14 
3.2.3.  Safety for the user  ................................ ................................ ................................ ............  18 
3.2.4.  Safety for the environment  ................................ ................................ ................................  18 
3.3. Efficacy  ................................ ................................ ................................ ............................  18 
3.3.1.  Chickens for fattening  ................................ ................................ ................................ ....... 19 
3.3.2.  Breeder hens  ................................ ................................ ................................ ....................  20 
3.3.3.  Efficacy in weaned pigs  ................................ ................................ ................................ ..... 20 
3.3.4.  Carcass and meat quality  ................................ ................................ ................................ ... 21 
3.4. Post-market monitoring  ................................ ................................ ................................ ..... 22 
4. Conclusions  ................................ ................................ ................................ ......................  22 
5. Recommendations  ................................ ................................ ................................ .............  23 
6. Remark  ................................ ................................ ................................ ............................  23 
Documentation prov ided to EFSA  ................................ ................................ ................................ .. 23 
References  ................................ ................................ ................................ ................................ ... 23 
Abbreviations  ................................ ................................ ................................ ...............................  25 
Appendix A – Studies on tissue deposition of GAA, creatine, creatinine and homocysteine in 
chicken for fattening given increasing levels of dietary GAA.  ................................ ................  26 
Appendix B – Effect of increasing doses of GAA on carcass and meat parameters of chickens for 
fattening ... ................................ ................................ ................................ .......................  33 
Appendix  C – Tables of GAA and related metabolite deposition in tissues/organs  ........................  34 
Annex - Executive Summary of the Evaluation Report of th e European Union Reference Laboratory for 
Feed Additives on the Method(s) of Analysis for Guanidinoacetic acid  ................................ ... 39 
 
  
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1. Introduction  
1.1. Background and Terms of Reference  
Regulation (EC) No 1831/20031 establishes rules governing the Community authorisation of additives 
for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person 
seeking authorisation for a feed additive or  for a new use of a feed additive shall submit an application 
in accordance with Article 7.  
The European Commission received a request from AlzChem AG (formerly AlzChem Trostberg GmbH)2 
for authorisation of the product guanidinoacetic acid  (GAA) , CreAMINO®, when used as a feed 
additive  for all animal species and categories  (category: nutritional; functional group: amino acids, 
their salts and analogues) . The original application was subsequently modified during the assessment 
to restrict the target species to chickens for fattening, chickens reared for breeding and pigs.3 
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) (author isation 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. The particulars and documents in support of the application 
were considered valid by EFSA as of 19 De cember  2012. 
According to Article 8 of Regulation (EC) No 1831/2003, 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 . EFSA shall deliver an opinion on 
the safety for the target animals, consumer, user and the environment and on the efficacy of the 
product GAA (CreAMINO®), when used under the proposed conditions of use (see Section 3.1. 6). 
1.2. Additional information  
The additive is currently authorised under the category nutritional additives, functional group amino 
acids, their salts and analogues, for chickens for fattening.4 The minimum and maximum dietary 
concentration is 600 mg GAA/kg comp lete feed.  
The Panel on Additives and Products or Substances used in Animal Feed ( FEEDAP ) of EFSA issued an 
opinion on the safety and efficacy of GAA for chickens for fattening (EFSA, 2009).  
2. Data and methodologies  
2.1. Data  
The present assessment is based on d ata submitted by the applicant in the form of a technical 
dossier5 in support of the authorisation request for the use of GAA (CreAMINO®) as a feed additive. 
The technical dossier was prepared following the provisions of Article 7 of Regulation (EC) No 
1831/2003 , Regulation E C No 429/2008  and the applicable EFSA guidance documents.  
The FEEDAP Panel use d the data provided by the applicant together with data from other sources , 
such as  previous risk assessments by EFSA and peer-reviewed scientific papers , to deliver the present 
output . 
EFSA has verified the European Union Reference Laboratory ( EURL ) report as it relates to the 
methods used for the control of the GAA (CreAMINO®) in animal feed. The Executive Summary of the 
EURL report can be found in  the Annex.6 
                                                           
1  Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in 
animal nutrition. OJ L 268, 18.10.2003, p. 29.  
2  AlzChem AG, Dr Albert -Franck -Strae, 32, D -83308 Trostberg, Germany.  
3  Technical  dossier/Other documentation/2015 -07-23 Change.  
4  Commission Reg. (EC) No 904/2009 of 28 September 2009/OJ L 256, 29.9.2009, p. 2. 
5  FEED dossier reference: FAD -2011-0043. 
6  The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep -FAD-2011-
0043-GAA.doc.pdf  
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2.2. Methodologies  
The approach followed by the FEEDAP Panel to assess the safety and the efficacy of GAA 
(CreAMINO®) is in line with the principles laid down in Regulation (EC) No 429/20087 and the relevant 
guidance documents : Guidance on nutritional additives (EFSA FEEDAP Panel, 2012 a), Guidance on 
zootechnical additives ( EFSA FEEDAP Panel, 2012b), Technical guidance: Tolerance and efficacy 
studies in target animals ( EFSA FEEDAP Panel, 2011) , Guidance for establishing the safety of additives 
for the c onsumer ( EFSA FEEDAP Panel, 2012c) and the  Guidance on studies concerning the safety of 
use of the additive for users/workers ( EFSA FEEDAP Panel, 2012d). 
3. Assessment  
The additive applied for contains GAA as active substance. GAA is a metabolic intermediate 
synthesised from the amino acids glycine and arginine. GAA is subsequently methylated to creatine. 
Creatine phosphate serves as a dynamic reservoir of high energy phosphate in exchange with 
adenosyl triphosphate (ATP).  
The additive is currently authorised under the category nutritional additives, functional group amino 
acids, their salts and analogues, for chickens for fattening.8 The minimum and maximum dietary 
concentration is 600 mg GAA/kg complete feed.  
The application of GAA covered its use in feed for  chickens for fattening, chickens reared for breeding 
and pigs. Chickens reared for breeding are not included as an animal category in Regulation EU 
429/2008. Upon request, the applicant defined this newly proposed category as ‘male and female 
birds reared  for breeding purposes (0 –65 weeks of age). GAA is intended to be supplemented as of 24 
weeks of age ’. The FEEDAP Panel interprets this definition as an intended use in breeder hens and 
roosters (from 24 weeks of age).  
The FEEDAP Panel reiterates that this  compound should not be considered as an amino acid, salt or 
analogue , although it shows a sparing action on arginine. Independently of whether it is endogenously 
produced or ingested, it cannot be converted to an amino acid. GAA is a precursor of creatine  and it is 
used as a substitute for dietary creatine.  
3.1. Characterisation  
3.1.1.  Manufacturing process9 
The production of GAA is based on the reaction of the amino  acid glycine with cyanamide in an 
aqueous solution.10 
3.1.2.  Characterisation of the active substance11 
Guanidinoacetic acid (International Union of Pure and Applied Chemistry (IUPAC) name: 2 -
(diaminomethylideneamino)acetic acid; synonyms: glycocyamine, guanidineacetic acid, guanidoacetic 
acid, guanidinoacetic acid, 2 -guanidinoacetic acid, 2 -([amino(imino)me thyl]amino)acetic acid, n-
(aminoiminomethyl)glycine, n-amidinoglycine) has the Chemical Abstracts Service (CAS) No 352 -97-6 
and European Inventory of Existing Commercial Chemical Substances (EINECS) No 206 -529-5. The 
chemical formula is C 3H7N3O2 and  the mo lecular weight is 117.1 Da. The structural formula is shown in 
Figure 1.  
                                                           
7  Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementati on of Regulation (EC) No 
1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and 
the assessment and the authorisation of feed additives. OJ L 133, 22.5.2008, p. 1.  
8  OJ L 256, 29.09.2009,  p. 2. 
9  This section has been amended following the confidentiality claims made by the applicant.  
10 Technical dossier/Section II.2.3.  
11 This section has been amended following the confidentiality claims made by the applicant.  
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Figure 1 : Molecular structure of guanidinoacetic acid  
GAA is a crystallised product (white crystalline powder) of at least 98% purity. The active substance 
contains by specification 1.5% of glycine,  0.5% dicyandiamide,  0.03% cyanamide,  0.002% 
melamine and ≤ 0.004% total triazines (melamine, ammeline, amme lide and cyanuric acid).12 
The analysis of five batches (ion chromatography) showed an average of 98.2% of GAA as is (range 
97.1–98.9% , one of the batches did not comply with the specification ). Other components were 
glycine (average 0.6%, range 0.4 –1.4%), dicyandiamide (average 0.09%, range 0.06 –0.18%), 
melamine (average 0.0006%, range 0.0002 –0.0011 %) and water (average 0.3%, range 0.1 –0.4%). 
Cyanamide was found below the limit of quantification ( LOQ, 0.0007%) in all five batches.13 On dry 
matter basis the a mount of identified substances is on average 99.2% (range 98.7 –99.5%) . 
The levels of heavy metals and arsenic were analysed in seven batches of GAA and in all of them lead 
and arsenic were lower than 1 mg/kg, cadmium lower than 0.07 mg/kg and mercury lower  than 0.04 
mg/kg.14 Considering the thresholds set for feedingstuffs in Directive 2002/32/EC,15 these 
concentrations are without any concern.  
The production of GAA is based on the reaction of the amino acid glycine with cyanamide in an 
aqueous solution ( Section 3.1.1). Regarding unavoidable side reaction products as melamine, 
ammeline, ammelide and cyanuric acid, these impurities were analysed in 40 batches  and the results 
are described in the technical dossier . 16  
The detected amounts of these impurities do not represent safety concerns.  
3.1.3.  Characterisation of the formulated additive  
The formulated additive CreAMINO® contains by specification 96% GAA, 1.5% of glycine,  0.5% 
dicyandiamide,  0.03% cyanamide,  0.002% melamine, 1% starch and 1% water. The 
specification is confirmed by the analysis of five batches, showing a mean of 98.1% GAA (range 98.0 –
98.3%).17 
CreAMINO® is a white to slight beige odourless granular additive. It has a pH of 8.1 at 10%  
(weight/weight)  in aqueous solution. Its solubility in water is 1.5 g/L at 15 C. The additive has a bulk 
density ranging from 400 to 750 kg/m3.18 
One batch of CreAMINO® was analysed by sieving and the percentage of particles with a diameter 
< 100 m was 2.7%; < 63 m were 1.8% and < 20 m was 0.7% . The particle size distribution of 
two more batches was analysed by laser diffraction and the fractions (v/v) below 50 and 10 m 
ranged between 3 –4% and 1 –2%, respectively.19 
                                                           
12 Technical dossier/Section II/Annex II.5 and supplementary information April 2014.  
13 Technical dossier/Supplementary information April 2014/Annex 1.2.  
14 Technical dossier/Supplementary information April 2014/Annex 1.3.  
15 Directive 2002/32/EC of the Europea n Parliament and of the council of 7 May 2002 on undesirable substances in animal feed. 
OJ L 140, 30.5.2002, p. 10.  
16 Technical dossier/Section II/Annex II.4 and Supplementary information April 2014/Annex 1.4.  
17 Technical dossier/Section II/Annex II.3.  
18 Technical dossier/Section II/Annex II.8.  
19 Technical dossier/Supplementary information November 2014/Annex SIn 2.1.  
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The dusting potential of three batches was determined using the Stauber –Heubach m ethod and varied 
between 3.5 and 7.1 g/m3.20 
3.1.4.  Stability and homogeneity  
The stability studies were made before restricting the animal species in the original application to pigs, 
chicken for fattening and breeder hens and roosters. The dataset is therefore p artially based on 
premixtures and complete feed for animal species which are no longer subject of the application 
(dogs, fish and crustaceans).  
Shelf life  
The stability of the active substance GAA in the formulated product CreAMINO® was studied in three 
batches under two environmental conditions (25 C at 60% relative humidity (RH) for 48 months, and 
40C at 75% RH for 12 months; packaging not described). Losses of up to 0.5% were observed after 
48 months storage at 25 C. No losses were detected after 12 months of storage at 40 C.21 No losses 
of GAA were observed in two additional batches of CreAMINO® stored at 25 C and 60% RH 
(packaging not described) for 24 months.  
Stability in premixtures  
The stability of GAA from one batch of the formulated additive (supplementation level 19.4%) was 
tested in three vitamin/mineral (without choline chloride) premixtures (starter, finisher and 
withdrawal, typical for chickens for fattening). The premixtures were packed in paper bags with  
polyethylene foil inlay, stored under three conditions: 45 C for 3 months, 60 C for 3 weeks and 85 C 
for 3 days.22 No losses of GAA were detected.  
Two granulated forms of the active substance (97.8% and 97.6% GAA), each supplemented at 
17.1% , were used to supplement a vitamin/mineral (containing choline chloride) premixture for dry 
dog food.23 Samples of approximately 100 g of each of the three premixtures were packed in double 
walled paper bags and stored under two different conditions (25 C and 65% RH for up to 15 months, 
or 40C and 75% RH for 6 months). No losses of GAA were detected.  
Stability in feedingstuffs  
The stability of GAA from one batch of the formulated additive supplemented at 600 mg/kg to a 
complete feed for chickens for fattening (based on w heat, soybean, maize and soybean oil) was 
studied.24 After mixing, the feed was conditioned and pelleted at two different temperatures (70 C or 
80C). The samples were packed in paper bags and stored at ambient temperature for 3 months. The 
mash feed (eight  samples, before conditioning) was analysed to contain 535 mg GAA/kg 
corresponding to 550 mg formulated additive/kg (519 –577 mg/kg). After pelleting at 70 C, the GAA 
content was 562 mg/kg and no losses were detected after 3 months. After pelleting at 80 C, the GAA 
content was 531 mg/kg (99.3% recovery) and no losses were detected after 3 months.  
The stability of one batch of the formulated additive when supplemented at 977 mg GAA/kg in a wet 
dog food (81% moisture, 8.5% crude protein  (CP), 5.0% fat, 2.5% as h and 0.5% crude fibre) was 
also tested.25 After heat sterilisation (127 C for 60 min) , the recovery of GAA was 90%. No additional 
losses were observed after storage at 25 C for 15 months or at 40 C for 6 months.  
A second study was performed to test the stability of the additive in a dry (92% of dry matter) dog 
food (based on poultry meat, maize, wheat and rice). Two batches of the active substance GAA (two 
granulated forms) were used to supplement a dry dog food at 10 ,700 mg/kg (1.07%). Processing 
condit ions were as follows: conditioning (100 C for 20 –30 s), extrusion (125 –130C for 5–10 s) and 
drying (50 C for 12 h). After extrusion, losses up to 3% were found. Feeds were packed in double 
walled paper bags and stored under two different conditions (25 C and 65% RH for up to 15 months, 
                                                           
20 Technical dossier/Supplementary information November 2014/Annex SIn 2.2.  
21 Technical dossier/Supplementary information April 2014/Annex 1.5 . 
22 Technical dossier/Supplementary information April 2014/Annex 1.6.  
23 Technical dossier/Supplementary information April 2014/Annex 1.20.  
24 Technical dossier/Supplementary information April 2014/Annex 1.7.  
25 Technical dossier/Supplementary  information April 2014/Annex 1.8.  
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or 40C and 75% RH for up to 6 months). Storage of samples collected before extrusion at 25 C and 
60% RH for 15 months or at 40 C and 75% RH for 6 months did not show any loss of GAA. Storage of 
extruded feed at 25 C and 6 0% RH for 15 months or at 40 C and 75% RH for 6 months resulted in 
losses of 4%.  
The applicant provided data on the stability in feed for fish (salmon and tilapia) and crustaceans 
(shrimps) during feed processing (conditioning, extruding, drying and coolin g). The losses did not 
exceed 4%.26 
Homogeneity  
The capacity to homogeneously distribute in final feed was studied for the formulated additive (one 
batch) in a poultry feed (based on wheat, soybean and maize; 600 mg GAA/kg) and a wet dog food 
(81% moisture,  8.5% CP, 5.0% fat, 2.5% ash and 0.5% crude fibre; 985 mg GAA/kg DM),27 and in 
two granulated forms in a mash dry dog food (based on poultry meat, maize, wheat and rice; 
2,200 mg GAA/kg). 28 The coefficients of variation ( 8–10 subsamples) were 4.8% at 600 mg  GAA/kg , 
4% at 985 mg/kg  and 2.3 –3.8% at 2 ,200 mg GAA/kg.  
3.1.5.  Physico -chemical incompatibilities in feed  
Physico -chemical incompatibilities or interactions with feed, carriers, other approved additives or 
medicinal products are not expected.  
3.1.6.  Conditions of use  
The additive is applied for use in feed for pigs, chickens for fattening and breeding hens and roosters  
(breeding hens and roosters  from 24 weeks of age onwards ). The formulated additive should be 
added to the final compound feed via premixture. No minimum  content is proposed. The maximum 
content proposed is 6 ,000 mg GAA/kg complete feed for pigs, and 3 ,000 mg GAA/kg complete feed 
for chickens for fattening and breeding hens and roosters.  
3.2. Safety  
The metabolic fate of GAA was described in a pre vious opinion (EFSA , 2009). It is summarised below.  
GAA is a metabolic intermediary product being synthesised from the amino acids glycine and arginine, 
with synthesis taking place mainly in the kidney and pancreas. After transport to the liver, GAA is 
methylated to cre atine. The formation of GAA from arginine and glycine is the principal regulated and 
rate-limiting step in the biosynthetic pathway of creatine. The formation of GAA and creatine cannot 
be reversed.  
Biological methylations and homocysteine formation are cl osely linked. Dietary GAA supplementation 
considerably increases the methylation demand and therefore induces hyperhomocystein aemia in 
animals.  
Available information on the absorption of GAA in the gut  is scarce . However, indirect evidence 
(Ostojic and Voj vodic-Ostojic, 2015) indicates that a high efficiency of absorption of GAA is likely  in 
mammals and birds . GAA as such can be excreted via the kidney  (Hoberman et al., 1948, Wyss and 
Kaddurah -Daouk, 2000) . 
3.2.1.  Safety for the target species  
Chickens for fatteni ng 
A total of five studies was provided, which investigated the potential adverse effects of GAA overdoses 
on performance and health status of chickens for fattening (35 –42 days of duration). Four of these 
studies (1 –4) were already assessed in a previous opinion (EFSA, 2009).  
                                                           
26 Technical dossier/Section II/Annex 1.21.  
27 Technical dossier/Supplementary information April 2014/Annex es 1.7 and 1.8 . 
28 Technical dossier/Supplementary information April 2014/Annex 1.20.  
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Studies 1 –3 
These trials were already part of the previous application.29 They were not considered as tolerance 
studies in the previous EFSA opinion (EFSA, 2009) due to deficiencies in design (lack of 
haematological and clinical -chemic al parameters).  
It should be noted that in these 42 -day studies with a sufficient number of animals and replicates , 
severe signs of intolerance were seen at about 8 ,000 mg GAA/kg feed (growth depression, more feed 
required per unit gain, increased mortalit y in two trials). In contrast, an equimolar dose of creatine 
monohydrate was well tolerated (about the same body weight and feed to gain ratio as the control 
group) in the two studies in which this comparison was made.  
Study 4  
This trial was already submit ted with the previous application and assessed (EFSA, 2009). The 
description was as follows . 
One-day-old male chickens for fattening (n=1 ,280; Ross 308) were assigned to five dietary treatments 
(eight replicates (floor pens) per treatment with 32 birds eac h).30 The basal pelleted diet 
(maize /wheat/soybean meal) was supplemented with 0, 600, 1 ,500, 3 ,000 or 6,000 mg GAA/kg feed. 
The experimental period of 0 –35 days was divided into a starter (0 –14 days) and a grower phase (15 –
35 days), and feed was available ad libitum . Production performance (day s 14 and 35) and some 
haematological characteristics (day 35; 10 birds/treatment) were recorded.31 Health and mortality 
were checked daily. The results of the trial are shown in Table 1. 
Table 1:  Effect of increasing dietary GA A fed for 35 days on performance, mortality and some 
haematological parameters of male broiler chicks (8 replicates x 32 birds/treatment)  
 Treatments  
GAA intended (mg/kg feed)  0 600 1,500 3,000 6,000 
GAA analysed, starter (mg/kg)  < 0.05  592 1,451  3,496  5,534  
GAA analysed, grower (mg/kg)  < 0.05  586 1,529 2,966 5,883 
Mortality (%)  5.5 1.2 3.5 4.3 6.6 
Weight gain (g/bird)  2,091a 2,094a 2,093a 2,061a 1,892b 
Feed intake (g/bird)  3,088a 3,110a 3,085a 2,976ab 2,792b 
Feed/gain ratio  1.48 1.49 1.48 1.47 1.48 
Leucocytes (G/L)  7.8ab 9.9a 8.0ab 7.6ab 3.9b 
MCV (fL)  133.6c 141.4bc 169.9ab 182.8a 184.5a 
MCH C (pg)1 44b 45b 54ab 58a 59a 
Different superscripts in a row indicate statistically significant differences (p ≤ 0.05) . 
GAA: guanidinoacetic acid; MCV: mean corpuscular volume; MCH : mean corpuscular haemoglobin  concentration . 
(1): The MCH values in the report were erroneously calculated with the absolute packed cell volume instead of percentage 
values. The given MCH values of 0.44, 0.45, 0.54, 0.58 and 0.59 f or the five groups, respectively, were therefore adjusted 
to obtain the correct dimension (pg).  
GAA concentrations up to 1 ,500 mg/kg feed did not affect performance and mortality of the chickens 
for fattening, whereas at 3 ,000 mg GAA/kg feed, body weight gain and feed consumption were 
slightly reduced, although the difference was not significant. At the highest inclusion level (6 ,000 mg 
GAA/kg feed), feed consumption was significantly impaired, resulting in significantly lower weight 
gains. An effect on fe ed to gain ratio was not observed and mortality was not affected by the GAA 
treatment.  
Serum substrates (total protein, albumin, cholesterol, glucose, urea, uric acid) and enzymes (ALT, 
AST, γ -GT) remained essentially unchanged.  
                                                           
29 Technical dossier/Section III /Annex III.2 and Annex III.4; Section IV/Annex IV.37/Part 3.  
30 Technical dossier/Section IV/Annex IV 49 and Section III/Annex III.8.  
31 Haematological parameters (leu cocytes, erythrocytes, haemoglobin, haematocrit, thrombocytes, mean corpuscular volume, 
mean corpuscular haemoglobin  concentration) and biochemical parameters (total protein, albumin, alanine aminotransferase, 
aspartae aminotransferase, gamma glutamyl transpeptidase, glucose, cholesterol, creatine, uric acid and urea (blood urea 
nitrogen) were determined.  
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Mean corpuscular volume  (MCV) of erythrocytes was significantly increased by 1 ,500 mg GAA/kg feed. 
Macrocytic erythrocytes are frequently seen in anaemia, which was not observed. Alternatively, a high 
MCV can be a sign of deficiency of vitamin B 12, folic acid and/or methyl donors (e xcept methionine). 
The basal diet contained 7.5 μg added vitamin B 12, 0.5 mg added folic acid/kg and 100 mg added 
choline -Cl/kg. Th ese supplementation levels could be considered to be marginally low, particularly for 
the methyl  donor choline. This conclusi on is supported by a recent study in rats, where 
hyperhomocystein aemia by GAA could be suppressed by dietary choline or betaine (Setoue et al., 
2008).  
Study 5  
This new trial does not meet the requirements of a tolerance study, but can be considered 
complem entary to the above tolerance study  (Study 4). A total of 85 1-day-old male chicks (Ross 308) 
was distributed to five groups with 17 birds each and fed for 35 day diets containing 0, 600, 1 ,200, 
3,000 or 6,000 mg CreAMINO® (97.6% GAA) /kg feed, respectively , with no replicates .32 The pelleted 
diets consisted mainly of maize, soybean meal and soybean oil, and were supplemented with L-lysine 
HCl, DL-methionine, L-threonine and L-valine and via premixture with 20 µg vitamin B 12, 1 mg folic acid 
and 460 mg cholin e chloride per kg diet. The starter diet (days 1 –21) contained 23.3% CP and 12.3 
MJ nitrogen -corrected apparent metabolisable energy (AME N)/kg, the grower diet (days 22 –35) 20.5% 
CP and 12.8 MJ AME N/kg. 
Body weight of the individual birds was recorded on d ays 21 and 35, and feed intake weekly on pen 
basis. Blood and tissue samples were taken at the end of the study from about 10 randomly selected 
birds per treatment. Blood samples were examined for haematology33 and clinical chemistry .34 Tissue 
samples (breas t muscle, liver, kidney) were analysed for GAA, creatine, creatinine and homocysteine, 
and organ weights (also including heart, thyroid gland and brain) were determined followed by 
pathological examination. All parameters investigated were subjected to a o ne-factorial ANOVA with 
the fixed effect of treatment , the individual animal being the statistical unit . Differences between 
treatments were assessed by Tukey test (Tukey –Kramer test if the number of samples was dissimilar). 
Table 2 summarises the results of the main parameters  measured . The performance data could not be 
assessed in detail because of the absence of replicates. However, they showed , in line with the other 
trials described above , a 32% reduction of body weight gain at about 6 ,000 mg CreAMINO®/kg feed.  
Table 2:  Effect of increasing dietary GAA fed for 35 days on some haematological parameters of 
male chickens for fattening  
 Treatments  
GAA intended (mg/kg feed)  0 586 1,171 2,928 5,856 
GAA analysed, starter (mg/kg)  10 602 1,185 2,752 5,843 
GAA analysed, grower (mg/kg)  1 579 1,147 2,728 5,495 
Leucocytes ( ln transformed /µL)1 10.4 9.9 10.0 10.2 10.0 
Mean corpuscular volume (fL)2 119 120 120 121 119 
Mean corpuscular haemoglobin  (pg)2 34 36 35 36 35 
(1): n=9 for all groups except 1 ,171 mg GAA/kg (n=7) . 
(2): n= 11, 12, 8, 10 and 10 (acceptable samples) for the five groups, respectively.  
The average mortality was 7.1% and was not related to the treatment  (0, 3, 2, 1 and 0 dead birds per 
treatment group, respectively) . The critical decline of leukocytes and in crease of MCV and MCH 
observed in study 4 (described above) as a consequence of increasing dietary GAA concentrations was 
not seen; no differences between the five groups were identified for leukocyte count, MCV and MCH. 
The absence of these adverse effect s is consistent with the explanation of the findings of the former 
                                                           
32 Technical dossier/Supplementary information November 2014/Annex SIn 2.3.  
33 Red blood cell count (erythrocytes, haemoglobin, haematocrit, MCV, MCH, mean corpus cular haemoglobin concentration 
(MCHC)), white blood cell count (thrombocytes, T  cells, B cells, leu cocytes, heterophils, monocytes, lymphocytes), and 
reticulocyte count.  
34 GAA, creatine, homocysteine, amino acids, cholesterol, total protein, albumin, aspartate aminotransferase (AST), alanine 
aminotransferase (ALT), gamma -glutamyl transfe rase (γ-GT), glutamate dehydrogenase (GLDH), lactate dehydrogenase 
(LDH), creatine kinase (CK) and uric acid.  
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study by the FEEDAP Panel , that the higher supplementation of the experimental diets with vitamin 
B12, folic acid and the methyl donor choline would have prevented these adverse effects  (EFSA, 2009) . 
No other significant differences between groups were found for the haematological parameters 
(except for a lower number of heterophils in the group with 586 mg GAA/kg compared to the control 
and lower erythrocyte count in the group with 2 ,928 mg  GAA/kg compared to the group with 1 ,171 
mg GAA/kg). Blood clinical chemistry showed some differences for cholesterol (a decrease in 
cholesterol of the group with 2 ,928 mg GAA/kg compared to all other groups except the 5 ,856 mg 
GAA/kg), triglycerides (a decrease at 1 ,171 and 5 ,856 mg GAA/kg), total protein (a decrease in  the 
two high GAA groups) and albumin (a decrease in the low and the two high GAA groups).  
The mean relative liver and kidney weights of the group with 1 ,171 mg GAA/kg (2.20 % and 0.589% 
of body weight, respectively) were slightly but significantly lower compared to control animals (2.56 % 
and 0.631% of body weight, respectively) and are considered to be incidental, because these changes 
were not seen at the next higher dose level (group with  2,928 mg GAA/kg). The gross lesions found in 
four animals (enlarged liver in the control group; heart adhered to pericardium, watery fluid in the 
abdomen and dark red focus in the thyroid, all in the group with 1 ,171 mg GAA/kg) were within the 
incidence o f background lesions that may be observed in chicken and could not be attributed to the 
treatment. Detected lesions and alterations are within the scope of the background that is reported in 
poultry at these ages, including coccidian manifestations. The le sions (inflammation, necrosis, fibrosis, 
telangiectasia) recorded in the liver, kidneys and skeletal muscle were distributed at random across 
treatments. In the group with 5 ,856 mg GAA/kg feed, the relative organ weights (3.14%  and 0.754 %, 
respectively) we re significantly higher compared with those of the control animals (2.74% and 0.65%, 
respectively).  
A dose 10-fold higher than the initially recommended dose of GAA for chickens for fattening (600 
mg/kg complete feed) is highly toxic to the target animals.  A maximum safe dose for chickens for 
fattening should therefore be established.  
In its previous opinion on the safety of GAA for chickens for fattening , the EFSA FEEDAP Panel 
concluded that safety of the applied dose range of 600 –1,200 mg GAA/kg feed for chicken for 
fattening was not demonstrated  (EFSA, 2009) . The lowest proposed dose, 600 mg GAA/kg feed, was 
considered safe, 1 ,200 mg GAA was not tested and 1 ,500 mg showed an increase in the MCV of 
erythrocytes which was considered as an adverse effect. Th e Panel hypothesised that this adverse 
effect would be probably the result of a deficiency in methyl groups (methyl donors) of the diet used 
in the study. The lack of effects on the haematological endpoints up to the highest dose tested (5 ,856 
mg GAA/kg fe ed) in a recent study where diets enriched with methyl donors were fed, supported the 
previous hypothesis. However, the adverse effect represented by depressed growth and feed intake 
was again evident at this highest dose. Owing to deficiencies of that stu dy (absence of replicates, low 
number of animals) , it is considered prudent not to take the next lower dose (2 ,928 mg GAA/kg feed) 
as safe level, but the second lowest dose (1 ,171 mg GAA/kg feed). The FEEDAP Panel considers 
therefore 1 ,200 mg GAA/kg comple te feed for chickens for fattening as safe, provided the diet is 
sufficiently supplemented with methyl group donors (e.g. choline chloride, vitamin B 12 and folic acid).  
Breeder hens  
No tolerance study was submitted for breeder hens and roosters. The only s tudy submitted examined 
the effect of graded levels of GAA below the maximum content recommended by the applicant on the 
fertility and hatchability of eggs.  
The effect of GAA supplementation on breeders of chickens for fattening was studied using 120 hens 
(Cobb 500) allotted to five dietary treatments (six replicates of four birds per treatment) with 
increasing leve ls of GAA (0, 400, 800, 1 ,200 or  1,600 mg GAA/kg feed).35 The intended concentrations 
of GAA were confirmed by analysis. The basal diet, mainly consisting of maize and soybean meal, was 
offered as mash. The study started with hens of an age of 50 weeks, eggs were collected after 
insemination at week 60. The eggs  were incubated to determine fertility, hatchability and embryo 
                                                           
 
35 Technical dossier/ Supplementary information April 2014/Annex 1.15.  
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mortality. No information is given concerning egg collection, the length of the collection period 
(estimated to be about 20 days), and on the time after insemination, factors that may influenc e 
fertility (Gumułka and Kapkowska, 2005). Also , no information was provided on the feeding regime of 
roosters. The measured parameters were analysed by ANOVA and a post hoc  multiple comparisons 
test (Tukey’s studentised range).  
The results are described in Table 3. A significant improvement in hatchability was observed for the 
groups with 800 and 1 ,200 mg GAA/kg feed in comparison with the control group and the group with 
1,600 mg GAA/kg feed. The differences in hatchability are the result of the different fertility rates as 
embryo mortality was not affected by the treatment.  
Table 3:  Effect of increasing dietary GAA in breeder hens for chickens for fattening fed for 10 weeks 
on reproductive parameters  (six replicates x four birds/treatmen t) 
 Treatments 
GAA intended (mg/kg feed)  0 400 800 1,200 1,600 
GAA analysed (mg/kg feed)  0 390 830 1,150 1,510 
Breeder performance       
Laying rate (%)  52.1 50.4 53.2 53.4 50.5 
Fertility (%)  80.3 85.2 96.5 96.4 81.8 
Hatchability (%)  65.7a 65.6a 83.8b 84.0b 65.0a 
GAA: guanidinoacetic acid.  
Different superscripts in a row indicate statistically significant differences at p<0.05 . 
The progeny of the hens fed different GAA levels, a total of 360 male chickens, was fed diets (starter, 
grower and finisher) not supplemented with GAA for 42 days. Group size was 72 birds (six replicates 
with 12 chickens each). No significant differences were seen concerning final body weight and feed 
intake ; however , feed to gain ratio for the groups derived from hens treated w ith 800 and 1 ,200 mg 
GAA/kg feed (1.44 and 1.48, respectively) was significantly higher than in groups from hens treated 
with 0 and 1 ,600 mg GAA/kg feed (1.61).  
In general, the study in breeder hens , albeit insufficient in terms of endpoints required for a  tolerance 
study, did not provide indications of intolerance up to 1 ,600 mg GAA/kg feed. However, the data do 
not allow a conclusion on the safe levels for breeder hens . As no data on roosters were submitted , no 
conclusion can be drawn . 
Weaned piglets  
The study with weaned piglets (33  days old, Piétrain x ACMC) was performed as a combined 
efficacy/tolerance study for 42 days.36 A total of 288 weaned piglets were distributed to six treatment 
groups (0, 600, 900, 1 ,200, 4 ,500 or 6,000 mg GAA/kg feed) resulting in eight pens with six pigs/pen 
(three of each gender) for each treatment. The basal diet contained maize, wheat, soybean meal, 
barley, skim milk powder (only pre -starter) and soybean oil. The diet was formulated as a pre -starter 
with 19.7% CP and 1.42% total lysine (first 2 weeks) and a starter with 20% CP and 1.3% total lysine 
(until day 42). Vitamin B 12 (0.03 mg/kg), choline chloride (300 mg/kg) and folic acid (1.7 mg/kg) were 
supplemented at somewhat higher levels than usua l to provide enough methyl donors and to avoid 
adverse effects on haematology. Zootechnical parameters studied were body weight, average daily 
gain (ADG), average daily feed intake (ADFI), feed to gain ratio (F/G) and mortality. At the end of the 
study , blood samples from 12 piglets per treatment were collected for haematology,37 and clinical 
blood biochemistry.38 Six pigs per treatment were necropsied, and the absolute and relative weight of 
the liver, spleen and kidneys determined. Data were analysed by ANO VA with dietary tre atment as 
source of variation. Initial body weight or final body weight was used as the covariate in the model for 
performance parameters and absolute organ weights, respectively. Pairwise comparisons were 
performed with the Tukey –Kramer  test. 
                                                           
36 Technical dossier/Supplementary information June 2015/Annex SIn 3.3.  
37 Red blood cell count (erythrocytes, haemoglobin, haematocrit, MCV, MCH and  mean corpuscular haemoglobin concentration 
(MCHC)), white blood cell count (thrombocytes and leukocytes) . 
38 Homocysteine, GAA, creatine, creatinine , triglycerides, cholesterol, total p rotein, albumin, aspartate aminotransferase (AST), 
alanine aminotransferase (ALT), gamma -glutamyl transferase (-GT), alkaline phosphatase and urea.  
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Two to 3 days after weaning, signs of diarrhoea (soft liquid faeces, dehydration, poor body condition) 
were observed in all experimental piglets that were treated with colistin given in drinking water for 
3 days. The Panel considers that this treatm ent had no effect on the outcome of the studies. Mortality 
was low and comparable among treatments (2 –4%). GAA did not affect performance parameters 
(means: 26.0 kg final body weight, 419 g ADG, 601 g ADFI, 1.43 F/G).  
Blood, organ and muscle tissue concent rations of GAA, homocysteine, creatine and creatinine are 
given in detail in the tissue deposition section ( Section 3.2.2). However, it should be noted here that 
(i) at dietary concentrations of 4 ,500 and 6 ,000 mg GAA/kg feed the GAA level in liver increas ed by 
factors of about 30 and 70 compared with 2 –6 mg/kg at the lower dietary GAA doses ; (ii) at these 
dietary GAA levels , creatinine in the liver and kidney was about 8 –15 and 5 times, respectively, above 
control level ; and (iii) homocysteine  in kidney was significantly above the control value at 6 ,000 mg 
GAA/kg feed.  
All organ weights except liver were comparable across treatments. An increase in the absolute and 
relative liver weight was seen in the groups supplemented with 900 and 6 ,000 mg GAA/kg feed (824 
and 818 g, 3.17 and 3.16%, respectively) compared to the untreated control (697 g, 2.17%). No 
histopathological correlate was found for this finding in the group with 6 ,000 mg GAA/kg feed (no 
histopathology was performed for the group of 9 00 mg GAA/kg). Although ANOVA (six treatments 
with six piglets each) indicated significant effect of the treatment for both parameters, a pairwise 
comparison by the Tukey –Kramer test (a post hoc  analysis with a single -step multiple comparison 
procedure) di d not find significant differences (p>0.05). It should be mentioned that the outcome of 
these statistics indicates the absence of evidence for differences, but it is incorrect to conclude from 
this result that there is evidence of absence of differences.  
The tolerance study on piglets fed diets enriched with methyl group donors did not show adverse 
effects up to the highest dose tested (6 ,000 mg GAA/kg feed) related to zootechnical parameters, 
haematology and clinical chemistry. Also , no adverse effects wer e identified by statistical evaluation of 
differences in necropsy with a small number of animals per group. In general , GAA appears to be 
tolerated by piglet s up to a dietary concentration of 6 ,000 mg/kg complete feed. However, 
considering the small number  of observations available for the critical endpoint liver weight, it is 
considered prudent to conclude on a lower GAA level than 6 ,000 mg/kg as tolerated by piglets and 
pigs for fattening. Taking into account the uncertainties mentioned above, the FEEDAP Panel 
concludes that 1 ,200 mg GAA/kg complete feed is safe for piglets and that this conclusion can be 
extended to pigs for fattening . 
Conclusion on the safety for the target species  
The safe level of the product appears to be correlated with the supply of  methyl donors.  
If an adequate supply of methyl  donors is provided  in the feed, a safe level of 1 ,200 mg GAA/kg 
complete feed could be established for chickens for fattening  and piglets . The conclusion on piglets 
can be extended to pigs for fattening.  
In the absence of adequate data, the FEEDAP Panel cannot conclude on the safety of GAA for breeder 
hens and roosters.  
3.2.2.  Safety for the consumer  
Tissue deposition  
In its former opinion on CreAMINO® (EFSA, 2009) , the FEEDAP Panel assessed the tissue deposition 
of GAA, its metabolites creatine and creatinine, and homocysteine based on data from four studies 
carried out on chickens for fattening. The Panel noted that feeding CreAMINO® to chickens in doses 
up to 6 ,000 mg GAA/kg feed resulted in a  dose-related decrease  of GAA concentrations in muscle 
tissue and except for the dose of 600 mg/kg feed ; the same was found in liver. On the other hand, 
the creatine and creatinine content s in muscle and liver were found to be consistently increased by 
GAA feed supplementation in those studies when analysed. Data on homocysteine were limited to 
plasma but showed the positive response to GAA supplementation to feed only at levels of 6 ,000 
mg/kg feed in chickens.  
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The present opinion is based on GAA deposition data from nine studie s with chickens for fattening 
including four studies previously submitted and assessed, and one study in piglets.  
Chickens for fattening  
Summary tables of the findings obtained in several studies in chickens for fattening are given in 
Appendi ces A and C  The volume of data allows to describe principles of tissue/organ deposition of 
GAA, creatine (and creatinine) and homocysteine for a feed supplementation rate between 200 and 
7,850 mg GAA/kg. The samples analysed w ere breast muscle, liver and kidney; plasma/ serum data 
were also available but to a lesser extent  (two studies) . 
Increasing dietary levels of GAA result ed in a lower content in breast muscle up to 6 ,000 mg GAA/kg 
feed. The concentration of GAA in muscle tissue of control animals ranges from 2 to 24 mg GAA/kg  
(see Table 1 7). A similar picture can be described for liver up to concentrations of 3,000 mg GAA/kg 
feed (lower than control values, which range from 23 to 161 mg GAA/kg , see Table 2 2). The GAA 
content of kidney reflects dietary concentration, p articularly at levels ≥ 3 ,000 mg GAA/kg feed, which 
is consistent with its excretion via this organ  (see Table 2 5). 
Breast muscle of control chickens showed creatine concentration ranging from about 4 ,000 to 5 ,000 
mg/kg  (see Table 1 8). Increasing dietary levels of GAA resulted in a corresponding increase in 
creatine in breast muscle which reflects the fact that creatine is the physiological methylation product 
of GAA. However , at levels of ≤ 6 ,000 mg GAA/kg feed , this increase would norm ally not exceed ca 
25%. The dose relation is much closer for creatine in liver; control values may range from 50 to 100 
mg creatine/kg, at 3 ,000 mg GAA/kg feed , creatine increase s to about 400 mg/kg and at ≥ 6 ,000 mg 
GAA/kg feed , about 1 ,000 mg creatine/kg  could be expected. Creatine in kidney (one study , see Table 
25) shows a positive correlation  with dietary GAA ranging from 65 mg/kg in the control group to 225 
mg/kg at 6 ,000 mg GAA/kg feed. Creatinine, the final catabolite of creatine, has a low concentr ation 
in the liver and kidney (about 1 mg/ kg) whereas a onefold increase could be expected in breast 
muscle in relation to the control group  (see Tables 19, 24 and 2 5). The tissue concentrations of 
creatinine do not respond to dietary GAA concentrations.  
The demethylation product of methionine, homocysteine, is expected to increase as a result of GAA 
intake. The few data available allow the conclusion that homocysteine in muscle, liver and kidney (as 
well as in plasma) will not increase up to GAA supplement ation of 3 ,000 mg/kg feed. For 6 ,000 mg 
GAA/kg feed, a significant elevation of homocystein e in muscle, kidney and plasma is described.  
Piglets  
In the tolerance study in piglets (see Section 3.2.1 for details of the experimental design) , blood, liver, 
kidneys and muscle tissue were analysed for GAA, creatine, creatinine and homocysteine.39 The 
results are summarised in Table 4. 
  
                                                           
39 Technical dossier/ Supplementary information April 2014/Annexes SIn 1.16 and SIn 1.17.  
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Table 4:  The influence of increasing dietary concentrations of GAA in piglet feed on plasma levels 
and deposition in liv er, kidney and muscle tissue of GAA, creatine, creatinine and 
homocysteine (12 piglets/treatment for plasma, six piglets/treatment for organs and 
tissue)  
 Treatments  
GAA intended (mg/kg feed)  0 600 900 1,200 4,500 6,000 
Analysed, pre -starter (mg/kg)  <1 727 996 1,230 4,480 6,040 
Analysed, starter (mg/kg)  <1 571 779 1,150 4,600 6,020 
GAA       
- in plasma (µmol/L)  8.0a 9.2a 9.4a 9.7a 27.7b 20.9ab 
- in liver (mg/kg)  2.2a 2.0a 3.5a 5.7a 117.2a 293.7b 
- in kidney (mg/kg)  154 131 133 89 134 135 
- in muscle (mg/kg)  <1.0 1.0 <1.0 <1.0 2.24 1.24 
Creatine        
- in plasma (mg/dL)  1.8a 2.5a 2.5a 2.2a 3.9b 4.3b 
- in liver (mg/kg)  130a 276ab 395ab 339ab 1,056b 1,940c 
- in kidney (mg/kg)  81a 146ab 215b 137ab 387c 378c 
- in muscle (mg/kg)  5,759 5,704 5,555 5,831 6,019 5,938 
Creatinine        
- in serum (µmol/L)  88.8 90.8 90.6 92.5 101.3  96.3 
- in liver (mg/kg)  10.3a 14.5a 12.2a 13.0a 44.8b 17.0a 
- in kidney (mg/kg)  73 63 67 58 116 56 
- in muscle (mg/kg)  41.0ab 78.7c 40.5a 45.8ab 80.5c 54.0b 
Homocysteine        
- in plasma (µ mol/L)  15.6a 15.5a 15.4a 19.6ab 20.2ab 23.1b 
- in liver (mg/kg)  1.47 - - - - 1.23 
- in kidney (mg/kg)  1.37a 1.69a 1.66a 2.28ab 1.93a 2.96b 
- in muscle (mg/kg)  0.37 - - - - 0.57 
GAA: guanidinoacetic acid.  
Values <1.0 mg GAA/kg suggest below limit of detection  (LOD) . 
Means with different superscript letters within a row are significantly different (p ≤ 0.05) . 
Plasma levels of creatine were only significantly higher  at the two highest supplementation levels 
(4,500 and 6 ,000 mg GAA/kg). Creatinine  in serum was not affected by GAA dietary supplementation 
(Table 4). In liver, GAA was significantly increased only in piglets fed diet enriched with 6 ,000 mg 
GAA/kg. However, considering the standard deviation, the ranges may be more informative; the rang e 
of the groups with 0 –1,200 mg GAA/kg feed was 1 –7 mg GAA/kg liver, the range for the group of 
4,500 mg GAA/kg feed was 8 –359 mg GAA/kg liver and that for the group 6 ,000 mg GAA/kg feed 93 –
492 mg GAA/kg liver. Homocysteine  in plasma showed a dose -dependent increase starting a t 1,200 
mg GAA/kg feed and reaching significance at 6 ,000 mg GAA/kg feed. In the higher dose group, a 
significant increase in homocysteine was also seen in the kidney. In the liver and muscle tissue , only 
values of the control group and the high -dose group were available which were not different.  
Toxicological studies  
Acute oral toxicity, mutagenicity and genotoxicity and repeated dose toxicity (28 - and 90 -day) studies 
have been submitted. All these stud ies had been already evaluated in a previous opinion (EFSA, 2009)  
where it was concluded that  “The mutagenicity/genotoxicity studies provided show sufficient evidence 
of the lack of any effects of concern in this respect. The effects reported in the 28 - and 90-day studies 
generally reflect physiological responses to high exposures to a metabolic intermediate and do not 
identify any novel or unexpected toxicity. Feeding CreAmino would not result in GAA, creatine or 
homocysteine concentrations in chicken tiss ues relevant to consumer safety.”  
An in vitro  gene mutation assay in Chinese hamster V79 cells was newly submitted. The potential of 
GAA to induce gene mutations at the HPRT locus of these cells in compliance with OECD guideline 476 
was examined. The exper imental design consisted in two separate experiments: in the first one , the 
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effect of GAA was analysed after a 4 -h incubation with and without metabolic activation (rat S9 mix); 
in the second experiment , two treatment times (4 and 24 h) were tested, withou t metabolic 
activation. Six concentrations of GAA  (37.5–1,200 µg/mL, the latter equivalent to 10 mM) were used 
and neither precipitation nor relevant cytotoxic effects occurred up to the maximum concentration. 
Results indicate that GAA did not induce gene mutations at the HPRT locus in V79 cells, while the 
positive controls performed as expected.  
A double blind study of GAA with placebo control (inulin) was conducted in 24 human volunteers (12 
male and 12 female), administering a daily dose of 2.4 g GAA ( Ostojic et al, 2013a).  GAA was given as 
a single bolus ( eight capsules of 300 mg) taken on one occasion each morning before breakfast for 
6 weeks. The dose was calculated to be equivalent to about 3 g creatine. The results are not divided 
by gender, but th e authors state that no gender differences were evident  in the parameters 
measured . Creatine, GAA and creatinine were analysed in both serum and urine samples  collected at 
baseline and 2, 4 and 6 weeks  after administration . Plasma homocysteine and serum ac tivities of 
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma -glutamyl 
transpeptidase and creatine kinase were also analysed.  
The expected increases in serum GAA, creatine and creatinine were found, together with urinary GAA . 
Self-reported clinical symptoms  (e.g. heartburn, intestinal cramping, diarrhoea, muscle cramping, 
bloatedness, weight gain, nausea)  were slightly higher in the GAA group, but the symptoms were mild 
and the difference was not statistically significant. Of  all the other biochemical markers measured , the 
only one affected was serum homocysteine, which was increased by about 20%. 
Hyperhomocystein aemia is associated with increased risk of cardiovascular disease and an increase in 
homocysteine is a direct conse quence of GAA ingestion. Ostojic et al. (2013b) investigated the 
addition of methyl donors (1.6 g/d ay of betaine HCl, 5 µg/day of vitamin B 12, 10 mg/day of vitamin B 6 
and 600 µg/day of folic acid) to administered GAA (2.4 g/day) in 20 human volunteers over 8 weeks 
and demonstrated that with this regime the previously observed elevation of homocysteine did not 
occur.  
Overall, the only concern from the human ingestion of  GAA is an increase in homocysteine which is 
unlikely to occur at the exposure levels from food of animal origin that might result from the use of 
GAA as a feed additive provided the maximum safe levels in feed concentration are not exceeded.  
Exposure to G AA, related metabolites and homocysteine  
The exposure assessment is made under the assumption that 1 ,200 mg GAA/kg feed (the highest 
dietary concentration of GAA which is considered safe) is the maximum content in complete feed for 
chickens for fattening a nd piglets/pigs for fattening.  
No additional exposure of the consumer to GAA from tissues and organs of treated animals is 
expected (see sections on tissue deposition in chickens for fattening and piglets  above). Although the 
creatine concentration in edib le tissues would increase in a dose -dependent manner as a result of GAA 
administration to target species, tissue concentrations would not essentially exceed background values 
reported in the literature (e.g . 5.26 g creatine/kg fresh be ef muscle (Mateescu e t al., 2015 ) or 3.7 g 
creatine/kg in pork muscle (Mora et al., 2008 )). Creatinine concentration is not substantially affected 
by dietary GAA. No additional exposure of the consumer to homocysteine resulting from the use of 
GAA in feed of chickens for fatte ning or piglets/pigs for fattening is expected.  
When the maximum concentrations considered safe in feed are exceeded, an increased exposure of 
the consumer to GAA from the consumption of pig liver at ≥ 4 ,500 mg GAA/kg feed and from the 
consumption of chick en liver at ≥ 6 ,000 mg GAA/kg feed should be expected. At these GAA levels in 
feed, creatine in meat from chickens for fattening would provide an additional 16 –25% creatine 
intake, and that of liver from chickens for fattening and piglets/pigs for fattenin g an increased intake 
by a factors of up to 19 and up to 15 , respectively. The same GAA levels in feed would also lead to an 
increase in homocysteine in meat  (up to 57%  in chicken for fattening , see Table 2 0), as also 
concluded from plasma data  (see Table 4 for pig and Table 29 for chicken for fattening) . This 
increase, which may be higher if the supplementation of the diet with methyl donors is low, would 
raise safety concerns for consumers.  
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Impurities  
The additive GAA is specified to contain impurities: d icyandiamide (≤ 0.5%), cyanamide (≤ 0.03%) 
and melamine (≤ 20 ppm). The first two were considered in the 2009 opinion on GAA. For 
dicyandiamide , the provisional tolerable daily intake (PTDI) was considered to be 1.0 mg/kg bw  per 
day (EFSA, 2009), while for  cyanamide the ADI was established as 0.002 mg/kg bw  per day (EFSA, 
2010). In 2009 , following a conservative scenario calculation for both impurities , the FEEDAP Panel 
concluded  that levels in tissues and organs  would result in daily intakes well below the  respective 
PTDI/ADI s and therefore without safety  concern s. 
The specified highest concentration of melamine in the additive is 20 ppm (mg/kg). This would result 
in levels of melamine in feed of 0.024 mg/kg at an inclusion rate of 1 ,200 mg GAA/kg. The maxi mum 
permitted level in feed (with the exception of the feed additives GAA, urea and biuret) is 2.5 mg 
melamine/kg (Directive 2002/32/EC),40 thus the contribution of GAA to melamine content in feed 
would be of no concern.  
Conclusions on the safety for the co nsumer  
In 2009, t he FEEDAP Panel concluded (EFSA, 2009):  ‘The mutagenicity/genotoxicity studies provided 
show sufficient evidence of the lack of any effects of concern in this respect. The effects reported in 
the 28 - and 90 -day studies generally reflect ph ysiological responses to high exposures to a metabolic 
intermediate and do not identify any novel or unexpected toxicity. Feeding CreAmino would not result 
in GAA, creatine or homocysteine concentrations in chicken tissues relevant to consumer safety ’. 
The newly submitted studies confirm the above conclusions. They are extended with respect to pork 
meat and liver consumption.  
There is no concern on consumer safety resulting from the use of GAA in feed for chickens for 
fattening and piglets/pigs for fattenin g provided the maximum GAA concentrations in feed considered 
safe (1,200 mg/kg feed) are respected.  
3.2.3.  Safety for the user  
The studies on skin and eye irritancy and on skin sensitisation submitted were already assessed in the 
former opinion (EFSA, 2009) and the new uses (new species) would not substantially modify the 
outcome of the assessment.41 GAA is not an irritant to eyes and skin and not a dermal sensitiser.  
The commercially available GAA is a granulated product containing up to 2% of re spirable particles 
and a dusting potential ranging between 4 and 7 g/m3 which is considered high. Significant inhalation 
exposure may occur. However , due to the absence of data on respiratory toxicity , no risk assessment 
can be performed.  
3.2.4.  Safety for the environment  
The safety for the environment was assessed in a previous opinion (EFSA, 2009) and the new uses 
(new species) would not modify the outcome of the assessment. GAA is a physiological molecule and 
occurs naturally in animals. GAA from CreAMINO® would not lead to a faecal excretion of molecules 
which are not normally present in the excreta of the target animals. The FEEDAP Panel concludes that 
the use of CreAMINO® as a feed additive is not expected to pose a risk to the environment.  
3.3. Efficacy  
The app licant provided a total of 10 studies on chickens for fattening,42 one study on breeders of 
chickens for fattening and one study on piglets. Out of them, seven studies on chickens for fattening 
have been already submitted with the previous submission and as sessed by the FEEDAP Panel (EFSA, 
2009). Concerning  the efficacy of CreAMINO® as feed additive for chickens for fattening, the former 
opinion concluded that ‘600 mg GAA kg/feed for chickens for fattening can be considered as the 
                                                           
40 DIRECTIVE 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on u ndesirable substances in animal 
feed. OJ L 140, 30.5.2002, p.  10. 
41 Technical dossier/Section III.3.3.1.1 and Annexes III.37, III.38 and III.40.  
42 Technical dossier/Section IV.  
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minimum dose improving perf ormance characteristics. However, the FEEDAP Panel notes that the 
effects are most consistently seen at 800 mg GAA/kg feed, which is in line with the dose level 
recommended by the applicant ’. 
A description of the studies supplied with the present applicati on is reported below.  
3.3.1.  Chickens for fattening  
The applicant provided a total of 10 studies on chickens for fattening. Out of them, seven studies on 
chickens for fattening ha d been already submitted with the previous submission and assessed by the 
FEEDAP Pan el (EFSA, 2009). Out of these seven,  four provided some evidence of efficacy and  two 
were rejected in the former assessment (EFSA, 2009) because of insufficient duration (21 days 
instead of the minimum of 35 days as required by Regulation 429/2008).43 One of the newly 
submitted  studies  was conducted between the 13 th and 27 th days of age;44 this study was not 
considered for the same reason.  The other two new submitted studies have been assessed.  
Study 1 
The study was carried out on 768 1-day-old male chick s (Ross 308) randomly allocated to four dietary 
treatment groups of six replicates, each containing 32 birds. A three-phase feeding scheme (starter, 
grower and finisher) was applied for three 13 -day periods of a total of 39 days.45 The basal diet 
mainly con sisted of maize and soybean meal (lysine/arginine ratio varied from 0.98 to 1.26). The four 
groups were fed the basal diet with 0, 600, 1 ,200 mg GAA/kg or the basal diet supplemented with fish 
meal (at 6% in the starter phase and at 3% in the growing and f inishing phases), respectively. 
Intended GAA concentrations in feed were confirmed by analysis. Feed was offered ad libitum  as 
mash. On day 26, samples of blood were taken from 12 birds per treatment to evaluate oxidative 
status. These same birds were killed to determine GAA, creatine and creatinine in breast  muscle . At 
the end of the trial, carcass and meat quality were assessed on 12 birds per treatment (except carcass 
analysis, six samples per treatment). Data were analysed by a linear model with the fix ed effect of 
treatment. The data of zootechnical performance were analysed considering the pen as statistical unit. 
Treatment means were compared by the post hoc Tukey test. Orthogonal contrast s were  applied to 
explore the effect of treatment. The results are summarised in Table 5. 
Mortality was low, ranged between 1.0 % and 3.1% and was not affected by treatment. The 
orthogonal contrasts detect ed an effect of the GAA treatment when compared with the control group.  
However, pairwise comparisons w ere not able to identify differences.  The treatments with GAA diets 
significantly improved final body weight, ADG and feed to gain ratio  compared with the group fed the 
basal diet . No significant differences were seen for these endpoints between the GAA -supplemented 
foods and the fish meal group.  
Table 5:  The influence of increasing dietary concentrations of GAA in chicken feed on growth 
parameters and mortality (192 chickens/treatment fed for 39 days). Positive control was 
supplemented with fish meal  
 Treatments  
Orthogonal contrast GAA 
diets vs negative control  GAA (mg/kg feed)  0 600 1,200  0 
Fish meal (%)  0 0 0 6/3 
Final body weight (g/bird)  2,639 2,707 2,695 2,706 p<0.05  
Average daily gain (g/d ay) 66.4 68.2 67.9 68.2 p<0.05  
Average daily feed intake 
(g/d ay) 108.9  110.3  108.6  110.5  NS 
Feed to gain  ratio  1.64 1.62 1.60 1.62 p<0.05  
Mortality  1.0 2.6 1.6 3.1 NS 
GAA: guanidinoacetic acid; NS: non -significant ( p>0.05). 
                                                           
43 Technical dossier/Section IV/Annex IV.32 and IV.34.  
44 Technical dossier/Section IV/Annex IV .35. 
45 Technical dossier/Section IV/Annex IV 45.  
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Study 2  
The trial lasted 36 days and was carried out on 540 1-day-old male chicks (Ross 308)  allotted to six 
dietary treatments ( nine replicates of 10 birds per treatment).46 Birds were fed a starter die t from day 
1 to 15, the grower diet from day 16 to 25 followed by the finisher until study conclusion. The basal 
diets consisted mainly of wheat, maize and soya bean meal. The basal diets were either fed as such 
or supplemented with 5% fish meal. These two types of diets were given with or without 600 mg 
GAA/kg feed. The six treatments were: no fishmeal and no GAA in all the three phases; no fish me al 
plus 600 mg GAA/kg in all the three phases; fish meal without GAA in all the three phases; fishmeal 
plus 600 mg GAA/kg in all the three phases; fishmeal only in the starter phase without GAA; fishmeal 
included only in the starter phase plus 600 mg GAA/k g in all the three phases. The diets were offered 
for ad libitum  access. The zootechnical parameters studied were body weight gain ( calculated per 
individual), feed consumption (per pen) and feed to gain ratio (per pen). Data were analysed by bi -
factorial ANOVA with fishmeal inclusion and GAA supplementation as the fixed effects  and the pen as 
statistical unit . 
Mortality rate was quite high (6.7 –8.9%), with no differences among treatments. Results for growth 
parameters indicate that the effects of fishmeal inclusion and GAA supplementation were of additive 
nature without interaction. GAA supplementation significantly improved final body weight, daily gain 
and feed to gain ratio.  
Table 6:  The influence of dietary supplementation of GAA and of fish meal in chicken feed  on 
production parameters (90 chickens/treatment fed for 36 days)  
 Treatments  Means  
GAA (mg/kg)  0 600 0 600 0 600 GAA 
and/or 
fishmeal  No GAA 
and/or 
fishmeal  Fishmeal (% diet)  0 0 5 5 5 (up to 
day 15)  5 (up to 
day 15)  
Final bw (g) 2,763 2,815 2,773 2,900 2,729 2,836 2,850b 2,755a 
ADG (g/d ay) 75.5 77.0 75.8 79.3 74.6 77.6 77.9b 75.3a 
ADFI (g/d ay) 119.6  119.3  119.2  121.2  117.9  120.1  120.2  118.9  
Feed to gain  ratio  1.58 1.55 1.57 1.53 1.58 1.55 1.54b 1.58a 
Mortality (%)  7.8 5.6 6.7 5.6 8.9 4.4 5.2 7.8 
GAA: guanidinoacetic acid; bw: body weight; ADG: average daily gain; ADFI: average daily feed intake.  
For final bw, ADG and feed to gain ratio , the means of the GAA -treated vs the non -GAA-treated groups were statistically 
different at p<0.001. The interaction fish meal with GAA was insignificant for all parameters.  
Considering the data previously assessed and the two new studies, the FEEDAP Panel concludes that 
GAA is efficacious in chickens for fattening at the minimum dose of 600 mg/kg.  
3.3.2.  Breeder hens  
The study is described in detail under Section 3.1.2. Although  it appears that 800 and 1 ,200 mg 
GAA/kg feed improved hatchability without a clear influence on embryo mortality , the duration of the 
study is too short and its description is insufficien t to allow conclusions on efficacy . 
3.3.3.  Efficacy in weaned pigs  
To investigate the efficacy of GAA (CreAMINO®) in weaned piglets, 336 4-week-old piglets (Piétrain x 
ACMC) were allotted, according to body weight and litter of origin, to four dietary treatments  each 
consisting of 14 replicates (pens) of six animals ( three males and three females).47 The experimental 
diets were supplemented with 0, 600, 900 or 1,200 mg CreAMINO®/kg feed (containing 97.7% GAA) 
corresponding to 586, 879 and 1 ,172 mg GAA /kg feed, re spectively (values confirmed by analysis). 
The study started at 33 days of age after a 5-day adaptation period and lasted 42 days. In the 
prestarter period from day 0 to day 14, the basal diet consisted of maize, wheat and soybean meal 
and skim milk powder , and in the starter period from 15  to 42 days of wheat, soybean meal, barley 
                                                           
46 Technical dossier/Section IV/Annex IV 46.  
47 Technical dossier/Supplementary information April 2014/Annex I.17.  
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and maize. Body weight (individual pigs) and feed intake (at pen level) were recorded on days 0, 14, 
28 and 42, F/G was calculated for the same periods. On the same days faeces w ere scored for 
consistency. Data analysis was conducted by ANOVA and taking the initial body weight as covariate , 
the dietary treatment as sources of variation  and the pen as statistical unit . 
Over the whole experimental period (0 –42 days), body weight and  weight gain were significantly 
improved in piglets receiving 1 ,200 mg GAA/kg feed when compared with unsupplemented animals 
(Table 9). Mortality was low and no differences among groups were detected. Faecal scores were 
unaffected by treatments.  
Table 7:  The influe nce of increasing dietary concentrations of GAA in piglet feed on growth 
parameters and mortality (84 piglets/treatment fed for 42 days)  
 Treatments  
GAA (mg/kg) intended  0 586 879 1,172 
GAA (mg/kg prestarter) analysed  <1 727 996 1,230 
GAA (mg/kg starter ) analysed  <1 571 779 1,150 
Initial body weight (kg)  8.51 8.30 8.30 8.04 
Final body weight (kg)  25.5a 26.0ab 26.3ab 27.3b 
Average daily gain (g/d ay) 406.7a 419.8ab 426.3ab 451.1b 
Average daily feed intake (g/d ay) 581.7  600.7  612.9  634.1  
Feed to gain  ratio  1.46 1.44 1.42 1.43 
Mortality (%)  2.4 2.4 3.6 2.4 
GAA: guanidinoacetic acid.  
a, b: Values in a row with different superscripts differ significantly (p ≤ 0.05) . 
 
3.3.4.  Carcass and meat quality  
In 2009, t he FEEDAP Panel assessed four studies in chickens for fattening for the influence of GAA on 
carcass and meat parameters. The Panel concluded that GAA when supplemented at  doses of ≥ 1 ,200 
mg GAA/kg complete feed reduces abdominal fat as noted in three trials. At 600 mg GAA/kg feed no 
consistent effects were noted for breast muscle yield (four trials), and no conclusions could be drawn 
regarding drip loss (two trials) or po st-mortem pH (one trial).  
Efficacy studies 1 and 2 included an assessment of the effect of the additive on carcass and/or meat 
quality, the results of which are summarised below. A third study (already submitted in the previous 
application) was not considered because the feeding time was too short (from d ay 14 to day 35).48 
For details of study design and conduct of the first efficacy study , see Section 3. 3.1.49 The main data 
from day 39 are given in Appendix B. The findings can be summarised as follows . 
Differences in carcass yield, carcass composition and carcass analysis were small and in most cases 
not significant. The absolute breast yield was significantly higher in the groups supplemented with 600 
and 1 ,200 mg GAA/kg feed compared with the control group fed purely vegetable diet.  
There were only minor effects of GAA supplementation on meat quality  detected basically by the 
orthogonal contrast (GAA diets versus negative control) . pH values in breast meat post mortem 
showed a tendency (p<0.1) to be lower than in the control group. Water binding capacity seemed to 
decrease in the groups treated with GAA diets  as a consequence of higher press losses (significant at 
1,200 mg GAA/kg feed , 18.3% in control versus 21.2% in 1 ,200 mg GAA/kg group, p<0.05)  and 
cooking losses . Differences in c olour (higher lightness and yellowness in GAA diets in relation to the 
control group) were marginal.  
Also, data on carcass quality were reported in the second  efficacy study  (Section 3.3.1) .50 At the end 
of the trial, six birds per pen were selected for car cass evaluation (three treatments each with and 
                                                           
48 Technical dossier/Section IV/Annex IV.34.  
49 Technical dossier/Section IV/Annex IV.45.  
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without GAA, 54 birds/treatment). For each bird, the weights of breast, legs and residual carcass were 
separately assessed. Carcass weight (control 1 ,845 vs GAA 1 ,925 g), dressing percentage (66.9 vs  
67.3%) and thigh weight (20.9 vs 21.2% of body weight) were significantly improved (p=0.009) by 
the addition of 600 mg GAA to complete feed. No differences were observed for relative breast weight 
(control 23.9% vs GAA 24.0%).  
Conclusions on product quali ty 
Percentage of breast meat was significantly improved by GAA (600 –1,200 mg/kg feed) in one study, 
and carcass yield (by 600 mg GAA/kg feed ) in another . The confirmation of th ese effect s would need 
more studies also in different animal species.  
Conclusions on efficacy  
Considering the data previously assessed and the two new studies, the FEEDAP Panel concludes that 
GAA is able to improve the performance  in chickens for fattening at the minimum dose of 600 mg/kg.  
In the absence of adequate data, th e FEEDAP Panel cannot conclude on the efficacy of GAA in 
breeder hens and roosters.  
In piglets, only one study was available  showing that 1,200 mg GAA/kg complete feed improved 
growth. When the assessment is based on zootechnical parameters, at least three  studies are 
required ; therefore , the FEEDAP Panel is not in a position to conclude on the basis of this single study.  
3.4. Post -market monitoring  
The FEEDAP Panel considers that there is no need for specific requirements for a post -market 
monitoring plan other  than those established in the Feed Hygiene Regulation51 and Good 
Manufacturing Practice.  
4. Conclusions  
The safe level of the product appears to be correlated with the supply of methyl donors. If an 
adequate supply of methyl donors is provided in the feed, a  safe level of 1 ,200 mg GAA/kg complete 
feed could be established for chickens for fattening and piglets. The conclusion on piglets can be 
extended to pigs for fattening. In the absence of adequate data, the FEEDAP Panel cannot conclude 
on the safety of GA A for breeder hens and roosters.  
GAA is not mutagenic. The effects reported in the 28 - and 90 -day studies do not identify any 
unexpected toxicity other than physiological effects. Feeding GAA would not result in GAA, creatine or 
homocysteine concentrations  in chicken and pig tissues/organs relevant to consumer safety provided 
the maximum GAA concentrations in feed considered safe are respected.  
GAA is not an irritant to eyes and skin and not a dermal sensitiser. Significant inhalation exposure may 
occur. Ho wever , due to the absence of data on respiratory toxicity , no risk assessment can be 
performed.  
The use of GAA in animal nutrition is not expected to pose a risk to the environment.  
Considering the data previously assessed and the two new studies, the FEEDAP Panel concludes that 
GAA is able to improve the performance in chickens for fattening at the minimum dose of 600 mg/kg. 
In the absence of adequate data, the FEEDAP Panel cannot conclude on the efficacy of GAA in 
breeder hens and roosters. In piglets , only one study was available showing that 1 ,200 mg GAA/kg 
complete feed improved growth. When the assessment is based on zootechnical parameters, at least 
three studies are required ; therefore , the FEEDAP Panel is not in a position to conclude on the bas is of 
this single study.  
                                                                                                                                                                                     
50 Technical dossier/Section IV/Annex IV.46.  
51 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements 
for fe ed hygiene. OJ L 35, 8.2.2005, p. 1.  
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5. Recom mendations  
The specification of the formulated additive should be modified to a minimum content of 98% GAA.  
The maximum safe levels of GAA in feed for chickens for fattening, piglets and pigs for fattening are 
derived under th e assumption that the feed contains sufficient amounts of methyl donors (other than 
methionine, e.g. choline, betaine and folic acid) and vitamin B 12. It is recommended to include the 
corresponding statement in other provisions.  
6. Remark  
In the view of the F EEDAP Panel, the functional group ‘amino acids, their salts and analogues’ 
describes substances which finally enter the metabolism of the body as amino acids and as such take 
part in the protein synthesis pathways. GAA does not play a comparable role. Alth ough resulting from 
the amino acid metabolism , GAA is exclusively converted to creatine and cannot be converted back to 
an amino acid. Grouping GAA under the functional group ‘amino acids, their salts and analogues’ 
would therefore not be consistent with t he logic behind thi s functional group. A solution could be that 
the definition of this functional group would be enlarged by the addition of ‘and related substances’ as 
is the case for the functional group ‘vitamins’ (chemically well -defined substances hav ing similar 
effect).  
Documentation provided to EFSA  
1. Guanidinoacetic acid (GAA) (CreAMINO®) for all animal species. March 2012. AlzChem 
Trostberg GmbH.  
2. Guanidinoacetic acid (GAA) (CreAMINO®) for all animal species. Supplementary information. 
April 2014. Su bmitted by AlzChem Trostberg GmbH . 
3. Guanidinoacetic acid (GAA) (CreAMINO®) for all animal species. Supplementary information. 
November 2014. Submitted by AlzChem Trostberg GmbH . 
4. Guanidinoacetic acid (GAA) (CreAMINO®) for all animal species. Supplementary in formation. 
July 2015. Submitted by AlzChem Trostberg GmbH . 
5. Evaluation report of the European Union Reference Laboratory for Feed Additives on the 
Methods(s) of Analysis for guanidinoacetic acid.  
6. Comments from Member States.  
References  
EFSA (European Food Safety Authority), 2009. Scientific Opinion of the Panel on Additives and 
Products or Substances used in Animal feed on a request of the European Commission on the 
safety and efficacy of CreAminoTM (guanidinoacetic acid) as feed additive for chickens for f attening. 
The EFSA Journal  2009, 988, 1 -30. 
EFSA (European Food Safety Authority), 2010. Conclusion on the peer review of the pesticide risk 
assessment of the active substance cyanamide. EFSA Journal 2010;8(11):1873. 61 pp. 
doi:10.2903/j.efsa.2010.1873  
EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2011. 
Technical guidance: Tolerance and efficacy studies in target animals. EFSA Journal 
2011;9(5):2175, 15 pp. doi:10.2903/j.efsa.2011.2175  
EFSA FEEDAP Panel (EFSA P anel on Additives and Products or Substances used in Animal Feed), 
2012a. Guidance for the preparation of dossiers for nutritional additives. EFSA Journal 
2012;10(1):2535, 14 pp. doi:10.2903/j.efsa.2012.2535  
EFSA FEEDAP Panel (EFSA Panel on Additives and P roducts or Substances used in Animal Feed), 
2012b. Guidance for the preparation of dossiers for zootechnical additives. EFSA Journal 
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Guanidinoacetic acid for chicken s for fattening, breeder hens and roosters and pigs  
 
 
 
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2012;10(1):2536, 19 pp. doi:10.2903/j.efsa.2012.2536  
EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 
2012c. Guidance for establishing the safety of additives for the consumer. EFSA Journal 
2012;10(1):2537, 12 pp. doi:10.2903/j.efsa.2012.2537  
EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 
2012d. Guidance on studies concerning the safety of use of the additive for users/workers.  EFSA 
Journal 2012;10(1):2539, 5 pp. doi:10.2903/j.efsa.2012.2539  
Gumułka M and Kapkowska E,  2005. Age effect of broiler breeders on fertility and sperm penetration 
of the perivitelline layer of the ovum. Animal Reproduction Science, 90, 135 –148. 
Hoberman HD, Sims EA and Peters JH, 1948. Creatine and creatinine metabolism in the normal male 
adult studied with the aid of isotopic nitrogen. Journal of Biological Chemist ry. 172, 45 -58. 
Mateescu RG, Garmyn AJ, O’Neil MA, Tait Jr RG, Abuzaid A, Mayes MS, Garrick DJ, Van Eenennaam 
AL, VanOverbeke DL, Hilton GG, Beitz DC and Reecy JM, 2015. Genetic parameters for carnitine, 
creatine, creatinine, carnosine and anserine concent ration in longissimus muscle and their 
association with palatability traits in Angus cattle. Journal of Animal Science, 90, 4248 -4255.  
Mora L, Sentandreu MA and Toldra F, 2008. Contents of creatine, creatinine and carnosine in porcine 
muscles of different metabolic types. Meat Science, 79, 709 -715. 
National Research Council (NRC), 1994. Nutrient requirements of poultry. 9th revised edition. National 
Academy Press, Washington, D.C.  
Ostojic SM, Niess B, Stojanovic M and Obrenovic M, 2013a. Creatine metabolism  and safety profiles 
after six -week oral guanidinoacetic acid administration in healthy humans. International Journal of 
Medical Sciences, 10, 141 -147. 
Ostojic SM, Niess B, Stojanovic M and Obrenovic M, 2013b. Co -administration of methyl donors along 
with guanidinoacetic acid reduces the incidence of hyperhomocysteinaemia compared with 
guanidinoacetic acid administration alone. British Journal of Nutrition, 110, 865 -870. 
Ostojic SM and Vojvodic -Ostojic A, 2015. Single -dose oral guanidinoacetic acid exhibits  dose-
dependent pharmacokinetics in healthy volunteers. Nutrition Research, 198 -205. 
Setoue M, Ohuchi S, Morita T and Sugiyama K,  2008. Hyperhomocysteinemia induced by 
guanidinoacetic acid is effectively suppressed by choline and betaine in rats. Bioscience, 
biotechnology and biochemistry, 72, 1696 -1703.  
Wyss M and Kaddurah -Daouk  R, 2000 . Creatine and creatinine metabolism. Physiological Reviews, 80,  
1107-1213.  
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Abbreviations  
 
ADFI average daily feed intake  
ADG average daily gain 
ADI average daily intake  
bw body weight 
CAS Chemical Abstracts Service  
EC European Commission  
EURL  European Union Reference Laboratory  
F/G feed to gain  ratio 
FEEDAP  Panel on Additives and Products or Substances used in Animal Feed 
GAA guanidinoacetic acid  
LOD limit of detection  
LOQ limit of quantification  
MCHC  mean corpuscular haemoglobin concentration  
MCV mean corpuscular volume  
PCr phosphocreatine  
PTDI provisional tolerable daily intake  
RH relative humidity  
 
 
  
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Appendix A – Studies on tissue deposition of GAA, creatine, creatinine and 
homocysteine in chicken for fattening given increasing levels 
of dietary GAA.  
Study 1 , in the 2009 opinion called Trial I: dose –response trial.  
At the end of study 1,52 samples of 114 chickens (three from each pen, 7 x 3 for both co ntrol groups, 
6 x 3 per treatment group) were killed for meat analysis. The results are shown in Table 8. 
Table 8:  The influence of increasing dietary concentrations of GAA in muscle GAA and creatine at 42 
days (18 chickens/group except the controls that had 21 chi ckens/group)  
 Treatments  
GAA intended (mg/kg)  0 314 628 942 1256  Fish meal  
GAA analysed, starter (mg/kg)  - 240 600 950 1100 - 
GAA analysed, grower (mg/kg)  - 200 6000 980 1400 - 
Creatine in muscle (mg/kg)  4665a 5337b 5370b 5322b 5689c 5215d 
GAA in muscle (mg/kg)  7.59b 1.30a 1.78a 1.21a 0.91a 1.41a 
GAA: guanidinoacetic acid.  
a, b, c, d  Values in a row with different superscripts differ significantly (p ≤ 0.05) . 
Creatine  content in breast muscle was significantly increased by GAA addition to feed at all doses and 
was higher than in a group given control diet containing fish meal. GAA content in breast muscle was 
significantly decreased by all doses of GAA in feed but was not different from the control diet 
containing fish meal.  
Study 2,  in the 2009 opinion named Trial IV: dose –response trial . 
On day 41, liver and muscle tissues were analysed from 120 chickens (five birds/pen; six 
pens/treatment).53 The results are summarise d in Table 9. 
Table 9:  The influence of increasing dietary concentrations of GAA in breast muscle and liver 
concentrations of GAA, creatine and creatinine at 41 days (30 chickens per treatment group)  
 Treatments  
GAA intended (mg/kg)  0 200 400 600 
GAA analysed, starter (mg/kg)  <0.08  158 338 499 
GAA analysed, grower (mg/kg)  <0.08  177 375 542 
GAA analysed, finisher (mg/kg)  <0.08  189 394 587 
Breast muscle      
- GAA (mg/kg)  23.7a 13.7b 6.2c 3.7c 
- Creatine (mg/kg)  3896c 4006c 4357b 4560a 
- Creatinine (mg/kg)  10.7b 13.0a 14.0a 14.5a 
- ATP (µmol/g 1h pm)  3.10b 3.31b 4.83a 4.30ab 
- AMP (µmol/g 1h pm)  0.81a 0.81a 0.57b 0.46b 
- IMP (µmol/g 1h pm)  4.06a 3.65ab 2.65b 2.67b 
Liver      
- GAA (mg/kg)  161.2b 215.0a 205.0ab 222.5a 
- Creatine (mg/kg)  45.3 52.0 48.8 54.2 
GAA: guanidinoacetic acid; ATP: adenosine triphosphate; AMP: adenosine monophosphate; IMP: inosine monophosphate . 
a, b, c   Values in a row with different superscripts differ significantly (p ≤ 0.05) . 
                                                           
52 Technical dossier/Section IV/Annex IV.37, part 2.  
53 Technical dossier/Section IV/Annex I V.42. 
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Creatine  content in breast muscle was significantly increased by GAA addition to feed at all doses and 
was higher than in a group given control diet containing fish meal. GAA content in breast muscle was 
significantly decreased by all doses of GAA in feed but was not different from the control diet 
containing fish meal.  
Study 3 , called in the former 2009 opinion Trial V: growth trial . 
On day 42, GAA, creatine and creatinine were analysed in breast meat from 48 chickens (three 
birds/pen; eight pens/treatment).54 The results are summarised in Table 10. 
Table 10:  The influence of increasing dietary concentrations of GAA in breast muscle GAA, creatine 
and creatinine at 42 days of age (24 chickens per treatment group)  
 Treatments  
GAA intended (mg/kg)  0 800 
GAA analysed, starter (mg/kg)  - 737 
GAA analysed, grower (mg/kg)  - 765 
GAA content (mg/kg)  1.81 0.70 
Creatine content (mg/kg)  4,481a 5,045b 
Creatinine content (mg/kg)  6.7 8.0 
GAA: guanidinoacetic acid.  
a,b Values in a row with different superscripts differ significantly (p ≤ 0.05) . 
GAA supplementation at 800 mg /kg feed significantly increased the creatine content of breast meat, 
but the reduction in GAA content was not significant and creatinine content was  essentially 
unchanged.  
Study 4 , for details see tolerance study 4 (in the 2009 opinion it was also addressed as tolerance 
study).  
The results of the biochemical analysis of breast muscle and liver (20 per treatment) and of the blood 
of male broiler chickens ( 10 per treatment) are summarised in Table 11.55 GAA concentration in breast 
muscle and liver decreased with increasing dietary GAA. There was also a dose -dependent increase of 
creatine and creatinine in the tissues and in plasma. A significant inc rease of plasma homocysteine 
was observed, but only in the highest GAA treatment group. High plasma homocysteine may be 
considered to be due to a reduced methyl group transfer capacity (either by a relative 
methionine/choline/betaine deficiency or a lack o f vitamin B12 and/or folic acid, or by both). The 
supplementation levels of those nutrients in the diet used could be considered to be marginally low, 
particularly for the methyl  donor choline.  
  
                                                           
54 Technical dossier/Section IV/Annex IV.43.  
55 Technical dossier/Section IV/Annex IV.49 and Section III/Annex III.8.  
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Table 11:  Effect of increasing dietary GAA on GAA, creatine, creatinin e and homocysteine 
concentrations in muscle, liver and plasma at day 36 (muscle and liver results from 20 
chickens/group, plasma values from 10 birds/group)  
 Treatments  
GAA intended (mg/kg feed)*  0 600 1,500 3,000 6,000 
GAA analysed, starter (mg/kg)  < 0.05 592 1,451 3,496 5,534 
GAA analysed, grower (mg/kg)  < 0.05  586 1,529 2,966 5,883 
Breast muscle       
- GAA (mg/kg)  3.95b 3.01b 1.26a 0.78a 0.85a 
- Creatine (mg/kg)  4,741a 5,157b 5,678c 5,863c 5,920c 
- Creatinine (mg/kg)  11.8a 12.3a 15.3ab 17.9bc 21.4c 
Liver       
- GAA (mg/kg)  45.0b 49.3b 47.3b 13.5b 6.2a 
- Creatine (mg/kg)  67.3c 90.0bc 113.2ab 114.2ab 132.4a 
Plasma       
- Creatine (mg/dL)  1.2a 1.1a 1.3ab 1.8b 2.6c 
- Creatinine (µmol/L)  3.6a 3.7ab 4.0ab 4.2b 4.2b 
- Homocysteine (µmol/L)  42.9a 39.1a 42.9a 39.6a 56.1b 
GAA: guanidinoacetic acid.  
a,b,c different superscripts within one row indicate significant differences (p ≤ 0.05) . 
Study 5, described in tolerance study 1.  
This study was already partly submitted and assessed in the 2009 opinion.  For the cur rent 
application, a new dataset with tissue deposition of GAA, creatine and creatinine data from chickens 
fed the unsupplemented diet, three diets supplemented with 785, 1 ,178 and 7 ,850 mg GAA/kg, 
respectively, was submitted.56 After 42 days of feeding GAA,  five birds per treatment (one per 
replicate) were slaughtered and liver and breast muscle analysed for GAA and creatine. Creatinine was 
measured in breast only.  
The deposition of GAA in muscle was not affected by GAA supplementation. In the liver, the hig hest 
dose of additive resulted in large accumulation of GAA. Creatine and creatinine deposition in muscle 
was increased already at the lowest GAA supplementation to feed. In the liver, the creatinine level 
was significantly increased only at the highest GA A dose (Table 12). 
Table 12:  The muscle and liver deposition of GAA, creatine and creatinine in chickens for fattening 
fed diets supplemented with increasing levels of GAA for 42 days (results of five chickens 
per treatment group)  
 Treatments  
GAA supplemented (mg/kg feed)  0 785 1,178 7,850 
GAA analysed, starter (mg/kg feed)  NA 690 1,090 6,600 
GAA analysed, grower (mg/kg feed)  NA 700 NA 7,750 
Breast muscle      
- GAA (mg/kg)  1.5ab 0.6a 0.4a 2.6b 
- Creatine (mg/kg)  4,051a 5,109b 5,192b 5,667b 
- Creatinine (mg/kg)  14.9a 23.6b 24.4b 34.0c 
Liver      
- GAA (mg/kg)  22.7a 14.6a 12.4a 103.9b 
- Creatine (mg/kg)  85.4a 131.6a 116.0a 1,667.5b 
                                                           
56 Techn ical dossier/Section III/Table III.5 and Annexes III.2 and III.10.  
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GAA: guanidinoacetic acid; NA: not analysed . 
a,b,c: means with different superscript letters within a  row are significantly different (p<0.05) . 
 
Study  6, details described under efficacy study 2. 
Samples of breast muscle ( two chickens/pen) were collected at the end of the experiment and 
analysed for GAA, creatine and creatinine.57 Results showed that regardless of fishmeal presence in 
feed, 600 mg GAA/kg feed resulted in the increased creatine content of muscle approximately by 12% 
(mean values of all groups ranged from 4 ,555 to 5 ,119 mg/kg, p<0.001). No effects on creatinine 
deposition were observed. Regardless of fishmeal presence in fee d, CreAMINO® supplementation led 
to significantly reduced content of GAA in breast (from 21.4 to 5.2 mg/kg).  
Study 7  
In a study on chickens for fattening , day-old chickens were fed for the first week basal diet based on 
maize, soybean and distiller's dried grains with solubles which was deficient in arginine (0.96 % 
arginine ).58 On day 8 post -hatch , 280 birds were allotted to seven groups each consisting eight pens 
(replicates) with five birds per replicate. Group 1 continued on the basal diet whereas diets for further 
six treatments were supplemented with 1.6 or 3.2 g arginine/kg (up to N ational Research Council  
requirements (NRC, 1994)) and/or 600 or 1 ,200 m g GAA/kg (see Table 5 for study design). 
Supplementations to feed were analytically confirmed. Analysed levels of folic acid, choline and 
betaine in diets were in the range of 0.9–1.1, 1 ,030–1,190 and 4 ,000–7,300 mg/kg, respectively.  
Chickens were fed thes e diets for a further 2 weeks up to their age of 22 days when one chicken per 
replicate was euthanised and samples of breast muscle were collected for analysis of creatine, 
homocysteine, phos phocreatine (PCr), adenosine triphosphate (ATP) and creatinine. B lood was 
collected from two additional birds from each replicate. Serum was analysed for GAA and creatine 
whereas homocysteine was measured in plasma.  
The deposition of homocysteine in muscle tended to be increased when a diet deficient in arginine 
and sup plemented with GAA was fed. However, upon the reaching of dietary arginine content 
complying NRC requirement (1.3%, by arginine dietary supplementation), both doses of GAA led to 
muscle homocysteine levels significantly lower and comparable with those foun d in birds of the 
negative control group. Total creatine and PCr deposition in breast showed a dose response to 
simultaneous GAA and arginine supplementation , whereas no response could be seen for free 
creatine. The muscle GAA concentrations were below det ection limit in the majority of samples and 
therefore not reported.59 Concerning blood derivatives, serum creatine also showed a dose response 
increase to simultaneous GAA and arginine supplementation , whereas GAA level was increased only in 
birds fed diets supplemented with GAA. The homocysteine concentration in plasma was not affected 
by GAA dietary treatments (Table 13). Serum creatinine levels were below detection limit and are not 
tabulated.60  In overall, the principal weakness of this study is the short duration ( 2 weeks only) of 
intake of diets supplemented with GAA.  
  
                                                           
57 Technical dossier/Section IV/Annex IV 46.  
58 Supplementary information November 2014/Annex SIn 2.8.  
59 LOD of GAA in muscle samples was 5 mg/kg.  
60 LOD of creatinine in serum was 83  µmol/L.  
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Table 13:  Deposition of homocysteine, total creatine, creatinine and phos phocreatine in breast 
muscle and plasma/serum levels of homocysteine, GAA and creatine in chickens for 
fattening fed diets supplemented with 600 or 1 ,200 mg GAA/kg and with different arginine 
concentrations for 2 weeks (results of eight chickens per treatment)  
 Treatments  
Total arginine (g/kg feed)  10.1  11.6  13.1  10.1  10.1  11.6  11.5  
Added GAA (mg/kg feed)  0 0 0 600 1,200 600 1,200 
Breast muscle         
- Homocysteine (nmol/kg as is)  75.3 108.6  119.0  114.3  82.7 78.3 70.9 
- Total creatine (g/kg as is)1 2.4 3.3 3.7 3.0 4.0 4.1 5.1 
- Creatinine (mg/kg as is)2 5.1 6.5 8.3 6.8 12.6 12.3 8.0 
- Phosphocreatine (mmol/kg DM)1 37.6 61.7 74.4 56.1 91.0 82.4 108.7  
Blood         
- Plasma homocysteine (µmol/L)  155.4  178.2  134.4  151.6  150.5  170.5  157.7  
- Serum GAA (µmol/L)1 0.39 0.48 0.91 3.85 9.84 4.91 13.95  
- Serum creatine (µ mol/L)1 14.2 19.8 28.3 22.3 37.1 26.0 43.2 
(1):  Linear response to graded dietary supplementation with GAA (mmol) in treatments 2, 6, and 7 ( p < 0.05) . 
(2): Linear response to graded dietary supplementation with arginine (mmol) in treatments 1, 2 and 3; and GAA ( mmol) in 
treatments 1, 4, and 5.  
Study 8 , described in detail under tolerance study 5.  
After 35 days (duration of the trial), samples of breast muscle, liver and kidney were collected from 10 
birds per treatment and analysed for GAA, homocysteine, creatine  and creatinine.61 
In the muscle and liver , GAA was significantly increased only by highest supplementation level 
whereas in kidney already dose 600 mg GAA/kg feed led to its increased deposition. No change was 
observed for creatine in muscle. In the liver and kidney , this compound deposition was significantly 
increased at supplementation levels 3 ,000 and 6 ,000 mg GAA/kg feed. Concerning creatinine, its 
increased level was found only in liver at highest GAA supplementation. A significantly higher 
concentrati on of homocysteine occurred only in the liver and kidneys also at highest GAA 
supplementation (Table 14). 
  
                                                           
61 Technical dossier/Supplementary information November  2014/Annex SIn 2.3.  
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Table 14:  Deposition of GAA, creatine, creatinine and homocysteine  in breast muscle, liver and 
kidney of chickens for fattening (Ross 308) fed diets supplemented with 0, 600, 3 ,000 or 
6,000 mg GAA/kg for 35 days (10 birds per treatment)  
 Treatments  
CreAMINO® added (mg/kg feed)  0 600 1,200 3,000 6,000 
GAA analysed, starter (mg/kg feed)  10 627 1,234 2,867 6,086 
GAA analysed, starter (mg/kg feed)  1 603 1,195 2,842 5,724 
Breast muscle       
GAA (mg/kg)  6 5 5 4 12(a) 
Creatine (mg/kg)  5,026 5,036 5,373 5,687 5,825 
Creatinine (mg/kg)  8.0 7.4 9.4 10.5 8.6 
Homocysteine  (mg/kg)  2.8 2.6 2.6 3.1 4.4(a) 
Liver       
GAA (mg/kg)  26 22 24 19 319(a) 
Creatine (mg/kg)  97 76 106 406(a) 893(a) 
Creatinine (mg/kg)  0.6 0.6 0.6 1.1 2.6(a) 
Homocysteine (mg/kg)  1.1 1.0 1.0 1.0 1.1 
Kidneys       
GAA (mg/kg)  27 50(a) 45 139(a) 253(a) 
Creatine (mg/kg)  65 73 91(a) 123(a) 225(a) 
Creatinine (mg/kg)  0.9 0.9 1.0 1.0 1.0 
Homocysteine (mg/kg)  1.6 2.0 1.6 1.9 2.4(a) 
GAA: guanidinoacetic acid.  
(a): Significantly different from birds fed unsupplemented diet (control) . 
Study 9  
The study, lasting 39 days, was carried out on 768 1-day-old male chicks (Ross 308) randomly 
allocated to four dietary treatment groups of six replicates, each containing 32 birds. A three-phase 
feeding scheme (starter, grower and finisher) was applied over three 13 -day periods for a total of 39 
days ( for a detailed description , see Section 3.3.1, chickens for fattening study 1).62 Treatment groups 
were: a negative control without GAA supplementation; a test diet in which GAA was added at 
600 mg/kg; a tes t diet in which GAA was added at 1 ,200 mg/kg and a positive control diet with no 
GAA addition and containing fish meal at 6% in the starter phase and at 3% in the growing and 
finishing phases. On day 26 of the trial, two birds/pen (12 per treatment) were e uthanized to 
determine GAA, creatine, creatinine and PCr/ATP ratio in breast muscle (Table 15). Dietary 
supplementation with GAA at both levels (600 and 1 ,200 mg/kg) resulted in reduced levels of GAA 
and increased levels of creatine in breast meat.  
Table 15:  Deposition of GAA, creatine, creatinine and phosphocreatine/ATP ratio in breast muscle of 
chickens for fattening (Ross 308) fed diets with increasing levels of GAA for 39 days (12 
birds per treatment)  
 Treatments  
Intended GAA (mg/kg feed)  0 600 1,200 Fish meal  
Analysed GAA, starter (mg/kg)  1 658 1,240 <1 
Analysed GAA, grower (mg/kg)  <1 575 1,196 <1 
Analysed GAA, finisher (mg/kg)  <1 545 1,190 <1 
GAA (mg/kg)  8.2b 2.2a 1.4a 5.9b 
Creatine (mg/kg)  4,789a 5,322b 5,541b 4,940a 
Creatinine (mg/kg)  5.5 6.0 6.1 5.7 
                                                           
62 Technical dossier/Section IV/Annex IV 45.  
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Phosphocreatine/ATP  2.4a 2.7ab 3.0b 2.6ab 
GAA: guanidinoacetic acid.  
a, b: Values in a row with different superscripts differ significantly ( p ≤ 0.05) .  
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Appendix B – Effect of increasing doses of GAA on carcass and meat 
parameters of chickens for fattening  
The study design is described in efficacy study 1. 
Table 16:  Effect of GAA supplementation on carcass and meat parameters in male chickens for 
fattening at slaughter (day 39; n=6 for carcass analysis, n=12 otherwise)1 
Item  Experimental diet2 SEM3 Significance4 
 NC (no 
GAA)  GAA 600 
mg/kg  GAA 1 ,200 
mg/kg  PC (fish 
meal 
6%)   I II 
Carcass composition  
Yield, %  
Breast meat, %  
Upper leg, %  
Lower leg, %  
Wing, %  
Rest, %  
Carcass analysis  
Moisture, %  
Crude protein, %  
Crude fat, %  
Breast meat  
pH, 0.5h  
1h 
2h 
3h 
4h 
24h 
T, 2h, °C  
3h, °C  
4h, °C  
Conductivity, μS  
Drip loss, %  
Press loss, %  
Thaw loss, %  
Cooking loss, %  
Shear force, N  
Colour  
Lightness  
Redness  
Yellowness  
Metmyoglobin %Δd0 -9 
TBARS [μg MDA/g meat]   
73.1 
29.4 
26.0 
13.3A  
10.3 
21.0 
 
66.1 
18.5 
12.8 
 
6.42 
6.33 
6.17 
6.04 
5.93B  
5.88B  
11.1B  
5.9 
2.9Bb  
7.81 
0.8 
18.3Aa  
3.5 
13.2A  
7.5 
 
51.1A  
6.1 
12.4A  
12.0 
1.46  
72.9 
30.4 
25.7 
13.0AB  
10.4 
20.5 
 
66.7 
18.6 
12.1 
 
6.31 
6.28 
6.10 
5.96 
5.81A  
5.76AB  
10.7AB  
6.1 
2.3ABab  
9.49 
1.0 
20.2ABab  
4.4 
14.6AB  
8.1 
 
53.0AB  
6.3 
13.6AB  
12.1 
1.50  
73.7 
30.7 
25.8 
13.2AB  
10.2 
20.0 
 
66.8 
18.5 
12.3 
 
6.39 
6.30 
6.07 
5.91 
5.83AB  
5.74A  
11.1B  
6.0 
2.5ABab  
8.74 
1.1 
21.2Bb  
4.4 
15.4B  
8.6 
 
53.5B  
6.9 
14.0B  
12.2 
1.38  
73.2 
30.2 
25.9 
12.6B  
10.4 
20.9 
 
65.9 
18.6 
13.0 
 
6.45 
6.34 
6.20 
6.01 
5.90AB  
5.81AB  
9.1A 
4.7 
1.7Aa  
8.98 
0.9 
19.3ABab  
4.4 
14.2AB  
8.5 
 
52.5AB  
6.4 
13.3AB  
12.5 
1.51  
0.25 
0.45 
0.29 
0.19 
0.12 
0.31 
 
0.34 
0.11 
0.40 
 
0.054  
0.059  
0.058  
0.043  
0.037  
0.038  
0.56 
0.45 
0.29 
0.586  
0.09 
0.70 
0.49 
0.61 
0.48 
 
0.66 
0.29 
0.44 
0.50 
0.090   
n.s. 
* 
n.s. 
n.s. 
n.s. 
T 
 
n.s. 
n.s. 
n.s. 
 
n.s. 
n.s. 
n.s. 
T 
* 
** 
n.s. 
n.s. 
n.s. 
T 
T 
** 
n.s. 
* 
n.s. 
 
* 
n.s. 
* 
n.s. 
n.s.  
n.s. 
n.s. 
n.s. 
* 
n.s. 
n.s. 
 
T 
n.s. 
n.s. 
 
n.s. 
n.s. 
n.s. 
n.s. 
T 
n.s. 
** 
* 
T 
n.s. 
n.s. 
n.s. 
n.s. 
n.s. 
n.s. 
 
n.s. 
n.s. 
n.s. 
n.s. 
* 
(1): Broilers were fed a corn -soybean starter diet from d ay 0 to day 13, a grower diet from d ay 14 to day 26 and a finisher diet 
from d ay 27 to day 39;  
(2): Values with different superscripts within a row are significantly different at P < 0.1 (A,B) or P < 0.05 (a,b)  Tukey test ;  
(3): SEM: standard error of least squares mean;  
(4): n.s.: non-significant ( P > 0.1); T : trend ( P < 0.1); * P < 0.05; ** P < 0.01; *** P < 0.001; I : orthogonal contrast of GAA -
supplemented diets vs. NC; II : orthogonal contrast of GAA -supplemented diets vs. PC; TBARS : thiobarbituric react ive 
substances; MDA : malondialdehyde.  
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Appendix C – Tables of GAA and related metabolite deposition in tissues/organs  
Chicken for fattening, breast muscle  
Table 17:  GAA in breast muscle for chickens for fattening (mg/kg muscle)  
Study  GAA in diet (mg/kg feed)  
Fish meal  Birds/  
treatment  Duration 
(days)  
0 200 314 400 600 800 942 1,200 1,500 3,000 6,000 7,850 
1 7.6b(*) - 1.3a - 1.8a - 1.2a 0.9a - - - - 1.4a(*) 18 or 21*  42 
2 23.7c 13.7b - 6.2a 3.7a - - - - - - - - 30 41 
3 1.8 - - - - 0.7 - - - - - - - 24 42 
4 4.0b - - - 3.0b - - - 1.3b 0.8a 0.9a - - 20 36 
5 1.5ab - - - - 0.6a - 0.4a - - - 2.6b - 5 42 
6 21.4b - - - 5.2a - - - - - - - - 18 35 
8 6a - - - 5a - - 5a - 4a 12b - - 10 35 
9 8.2b - - - 2.2a - - 1.4a - - - - 5.9b 12 39 
a,b,c Different superscripts within one row indicate significant differences (p ≤ 0.05).  
* Groups with 21 birds instead of 18.  
Table 18:  Creatinine  in breast muscle of chickens for fattening (mg/kg muscle)  
Study  GAA in diet (mg/kg feed)  Fish 
meal  Birds/  
treatment  Duration 
(days)  
0 200 314 400 600 800 942 1,200 1,500 3,000 6,000 7,850 
1 4,665a(*) - 5,337b - 5,370b - 5,322b 5,689c - - - - 5,215d 18 (21*)  42 
2 3,896a 4,006
a - 4,357b 4,560c - - - - - - - - 30 41 
3 4,481a - - - - 5,045b - - - - - - - 24 42 
4 4,741a - - - 5,157b - - - 5,678c 5,863c 5,920c - - 20 36 
5 4,051a - - - 5,109b - - 5,192b - - - 5,667b - 5 42 
6 4,558a - - - 5,119b - - - - - - - - 18 35 
7(1) 3,300 - - - 4,100 - - 5,100 - - - - - 8 22 
8 5,026 - - - 5,036 - - 5,373 - 5,687 5,825 - - 10 35 
9 4,789a - - - 5,322b - - 5,541b - - - - 4,940a 12 39 
(1): Linear response to graded dietary supplementation with GAA (p ≤ 0.05) . 
a,b,c,d  Different superscripts within one row indicate significant differences (p ≤ 0.05).  
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* Groups with 21 birds instead of 18.  
 
Table 19:  Creatinine in breast muscle of chickens for fattening (mg/kg muscle)  
Study  GAA in diet (mg/kg feed)  Fish 
meal  Birds/  
treatment  Duration  
(days)  0 200 400 600 800 1,200 1,500 3,000 6,000 7,850 
2 10.7a 13.0b 14.0b 14.5b - - - - - - - 30 41 
3 6.7 - - - 8.0 - - - - - - 24 42 
4 11.8a - - 12.3a - - 15.3ab 17.9bc 21.4c - - 20 36 
5 14.9a - - 23.6b - 24.4b - - - 34.0c - 5 42 
6 6.6 - - 6.9 - - - - - - - 18 35 
7 6.5 - - 12.3 - 8.0 - - - - - 8 22 
8 8.0 - - 7.4 - 9.4 - 10.5 8.6 - - 10 35 
9 5.5 - - 6.0 - 6.1 - - - - 5.7 12 39 
a,b,c Different superscripts within one row indicate significant differences (p ≤ 0.05).  
Table 20:  Homocysteine  in breast muscle of chickens for fattening (R7 nmol/kg muscle as is and R8 mg/kg muscle)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 600 1,200 3,000 6,000 
7 108.6  78.3 70.9 - - 8 22 
8 2.8a 2.6a 2.6a 3.1a 4.4b 10 35 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.0 5). 
Table 21:  Phosphocreatine in breast muscle (study 7 nmol/kg muscle DM and study 9 phosphocreatine/ATP)  
Study  GAA in diet (mg/kg feed)  Fish 
meal  Birds/  
treatment  Duration 
(days)  0 600 1,200 
7(1) 61.7 82.4 108.7  - 8 22 
9 2.4a 2.7ab 3.0b 2.6ab 12 39 
(1): Linear response to graded dietary supplementation with GAA (p ≤ 0.05) . 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.0 5). 
 
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Chicken for fattening, liver  
Table 22:  GAA in liver of chickens for fattening (mg/kg liver)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 200 400 600 1,200 1,500 3,000 6,000 7,850 
2 161.2a 215.0b 205.0ab 222.5b - - - - - 30 41 
4 45.0b - - 49.3b - 47.3b 13.5b 6.2a - 20 36 
5 22.7a - - 14.6a 12.4a - - - 103.9b 5 42 
8 26a - - 22a 24a - 19a 319b - 10 35 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.05).  
Table 23:  Creatine in liver of chickens for fattening (mg/kg liver)  
Study  GAA in diet  (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 200 400 600 1,200 1,500 3,000 6,000 7,850 
2 45.3 52.0 48.8 54.2 - - - - - 30 41 
4 67.3a - - 90.0ab - 113.2bc 114.2bc 132.4c - 20 36 
5 85.4a - - 131.6a 116.0a - - - 1667.5b 5 42 
8 97a - - 76a 106a - 406b 893b - 10 35 
a,b,c Different superscripts within one row indicate significant differences (p ≤ 0.05).  
Table 24:  Study 8 : Creatinine and homocysteine in liver of chickens for fattening (10 birds/treatment and 35 days of duration)  
Parameter  GAA in diet (mg/kg feed)  
0 600 1,200 3,000 6,000 
Creatinine (mg/kg liver)  0.6a 0.6a 0.6a 1.1a 2.6b 
Homocysteine (mg/kg liver)  1.1 1.0 1.0 1.0 1.1 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.05).  
 
 
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Chicken for fattening, kidney  
Table 25:  Study 8 : GAA, creatine, creatinine and homocysteine in kidney of chickens for fattening (10 birds per treatment and 35 days duration)  
Parameter  GAA in diet (mg/kg feed)  
0 600 1,200 3,000 6,000 
GAA (mg/kg kidney)  27a 50b 45ab 139b 253b 
Creatine  (mg/kg kidney)  65a 73a 91b 123b 225b 
Creatinine (mg/kg kidney)  0.9 0.9 1.0 1.0 1.0 
Homocysteine (mg/kg kidney)  1.6a 2.0a 1.6a 1.9a 2.4b 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.05).  
Chicken for fattening, blood  
Table 26:  GAA  in serum of chickens for fattening (µmol/L)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 600 1,200 
71 0.48 4.91 13.95  8 22 
(1): Linear response to graded dietary supplementation with GAA (p ≤ 0.05) . 
Table 27:  Creatine  in serum of chickens for fattening (R4 in mg/dL and R7 in µmol/L)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 600 1,200 1,500 3,000 6,000 
4 1.2a 1.1a - 1.3ab 1.8b 2.6c 20 36 
71 19.8 26.0 43.2 - - - 8 22 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.05).  
(1): Linear response to graded dietary supplementation with GAA (p ≤ 0.05) . 
 
 
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Table 28:  Creatinine in plasma of chickens for fattening (µmol/L)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 600 1,500 3,000 6,000 
4 3.6a 3.7ab 4.0ab 4.2b 4.2b 20 36 
a,b different superscripts within one row indicate significant differences (p ≤ 0.05).  
 
 
Table 29:  Homocysteine in plasma of chickens for fattening (µmol/L)  
Study  GAA in diet (mg/kg feed)  Birds/  
treatment  Duration 
(days)  0 600 1,200 1,500 3,000 6,000 
4 42.9a 39.1a - 42.9a 39.6a 56.1b 20 36 
7 178.2 170.5 157.7  - - - 8 22 
a,b Different superscripts within one row indicate significant differences (p ≤ 0.05).  
 
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Annex - Executive Summary of the Evaluation Report of the European 
Union Reference Laboratory for Feed Additives on the Method(s) of 
Analysis for Guanidinoacetic acid  
 
Guanidinoacetic acid (GAA)  is already authorised as feed a dditive for chickens for fattening. In the 
current application authorisation is sought under Article 4(1) for guanidinoacetic acid  under the 
category/functional group 3(c) 'nutritional additives'/'amino acids, their salts and analogues', 
according to Annex  I of Regulation (EC) No 1831/2003. Authorisation is sought for all animal species . 
For the determination of GAA in the feed additive and feedingstuffs  the Applicant proposed a single 
laboratory validated and further verified method based on Ion Chromatogr aphy coupled to UltraViolet 
detection (IC -UV). The performance characteristics recalculated by EURL, based on the experimental 
data provided by the Applicant, are:  
 a relative standard deviation for repeatability (RSDr) ranging from 0.11 to 4.2%;  
 a recovery  rate (R Rec) ranging from 96.4 to 99.1%; and  
 a limit of quantification (LOQ) in the feedingstuffs  of 50 mg/kg.  
For the determination of GAA in premixtures  the Applicant proposed a single laboratory validated 
method based on High Performance Liquid Chromatography coupled to UltraViolet (HPLC -UV), which 
is very similar to the method applied for the feed additive and feedingstuffs. The Applicant reported 
RSDr values ranging from 0.6 to 1.4% and R Rec values ranging from 96 to 108%.  
Based on the performance characteristics presented, the EURL recommends for official control (1) the 
single laboratory validated and further verified method based on ion chromatograph y coupled to 
ultraviolet detection to determine guanidinoacetic acid in the feed additive and feedingstuffs  and (2) 
the single laboratory validated to determine guanidinoacetic acid  in the premixture . 
Further testing or validation of the methods to be performed through the consortium of National 
Reference Laboratories as specified by article 10 (Commission Regulation (EC) No 378/2005) is not 
considered necessary.  
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