SCIENTIFIC OPINION
ADOPTED: 28 September 2022
doi: 10.2903/j.efsa.2022.7269
Safety and ef ﬁcacy of a feed additive consisting of
guanidinoacetic acid for all animal species (Alzchem
Trostberg GmbH)
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP),
Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik Christensen,
Birgit Dusemund, Mojca Fa /C20smon Durjava, Maryline Kouba, Marta L /C19opez-Alonso,
Secundino L /C19opez Puente, Francesca Marcon, Baltasar Mayo, Alena Pechov /C19a, Mariana Petkova,
Fernando Ramos, Yolanda Sanz, Roberto Edoardo Villa, Ruud Woutersen, J €urgen Gropp,
Montserrat Anguita, Jaume Galobart, Jordi Ortu ~no Casanova, Fabiola Pizzo and
Jordi Tarr /C19es-Call
Abstract
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 scienti ﬁc opinion on the safety and
efﬁcacy of guanidinoacetic acid (GAA) when used as nutritional additive in feed and water for drinking
for all animal species. The FEEDAP Panel concluded that GAA at 1,200 mg/kg complete feed was safefor chickens for fattening, chickens reared for breeding/laying, piglets and pigs for fattening. Thisconcentration in complete feed would correspond to maximum concentrations in water of 600 mgGAA/L. The Panel was not in a position to conclude on a safe level of GAA in laying/reproductive birds.In the absence of data on ruminants and salmonids, the FEEDAP Panel could not conclude on thesafety of GAA for all animal species. There was no concern on consumer safety resulting from the useof GAA in feed for poultry and pigs at the proposed conditions of use. The limited data did not allow to
conclude on the safety for the consumer when the additive was used in feed for ruminants or ﬁsh.
GAA was not toxic by inhalation, it was not an irritant to skin and eyes, and it was not a dermalsensitiser. The FEEDAP Panel concluded that the use of GAA as feed additive was not expected to posea risk to the environment. The use of the additive under assessment in animal nutrition at theproposed conditions of use had the potential to be ef ﬁcacious in all growing avian, Suidae and
ruminant (except for preruminants) species; in growing ﬁnﬁsh other than salmonids and in frog. It
was not possible to conclude on the ef ﬁcacy of the additive in other species, and in reproductive
animals.
©2022 Wiley-VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.
Keywords: nutritional additive, guanidinoacetic acid, safety, ef ﬁcacy, homocysteine, all animal species
Requestor: European Commission
Question number: EFSA-Q-2020-00521
Correspondence: feedap@efsa.europa.eu
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Panel members: Vasileios Bampidis, Giovanna Azimonti, Maria de Lourdes Bastos, Henrik
Christensen, Birgit Dusemund, Mojca Fa /C20smon Durjava, Maryline Kouba, Marta L /C19opez-Alonso, Secundino
L/C19opez Puente, Francesca Marcon, Baltasar Mayo, Alena Pechov /C19a, Mariana Petkova, Fernando Ramos,
Yolanda Sanz, Roberto Edoardo Villa and Ruud Woutersen.
Declarations of interest: The declarations of interest of all scienti ﬁc experts active in EFSA ’s work
are available at https://ess.efsa.europa.eu/doi/doiweb/doisearch.
Acknowledgments: The Panel wishes to thank the following for the support provided to this
scienti ﬁc output: Martina Reitano, the experts of the Animal Nutrition WG and the experts of the
Toxicology WG.
Erratum: The data provided allow to extend the safety of chickens for fattening to chickens reared for
breeding/laying and it was not done. It has been corrected in this version. To avoid confusion, the
original of the output has been removed from the EFSA Journal, and it remains available on request.
Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used
in Animal Feed), Bampidis V, Azimonti G, Bastos ML, Christensen H, Dusemund B, Fa /C20smon Durjava M,
Kouba M, L /C19opez-Alonso M, L /C19opez Puente S, Marcon F, Mayo B, Pechov /C19a A, Petkova M, Ramos F , Sanz
Y, Villa RE, Woutersen R, Gropp J, Anguita M, Galobart J, Ortu ~no Casanova J, Pizzo F and Tarr /C19es-Call J,
2022. Scienti ﬁc Opinion on the safety and ef ﬁcacy of a feed additive consisting of guanidinoacetic acid
for all animal species (Alzchem Trostberg GmbH). EFSA Journal 2022;20(5):7269, 17 pp. https://doi.
org/10.2903/j.efsa.2022.7269
ISSN: 1831-4732
©2022 Wiley-VCH Verlag GmbH & Co. KgaA on behalf of the European Food Safety Authority.
This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License,
which permits use and distribution in any medium, provided the original work is properly cited and nomodi ﬁcations or adaptations are made.
The EFSA Journal is a publication of the European Food Safety
Authority, a European agency funded by the European Union.
Guanidinoacetic acid for all animal species
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Table of contents
Abstract ............................................................................................................................... .................... 1
1. Introduction ............................................................................................................................... .4
1.1. Background and Terms of Reference ............................................................................................. 4
1.2. Additional information .................................................................................................................. 4
2. Data and methodologies .............................................................................................................. 4
2.1. Data ............................................................................................................................... ............ 4
2.2. Methodologies ............................................................................................................................. 4
3. Assessment ............................................................................................................................... .. 5
3.1. Characterisation .......................................................................................................................... 5
3.1.1. Characterisation of the guanidinoacetic acid crystalline powder ....................................................... 5
3.1.2. Characterisation of the formula ted additive .................................................................................. 5
3.1.3. Stability and homogeneity ............................................................................................................ 6
3.1.4. Conditions of use ........................................................................................................................ 6
3.2. Safety ............................................................................................................................... ......... 6
3.2.1. Absorption, distribution, metabolism and excretion ........................................................................ 7
3.2.2. Toxicological studies .................................................................................................................... 7
3.2.3. Safety for the target species ........................................................................................................ 8
3.2.3.1. Laying hens ............................................................................................................................... .8
3.2.3.2. Fish............................................................................................................................... ............. 9
3.2.3.3. Conclusions on safety for the target species .................................................................................. 9
3.2.4. Safety for the consumer .............................................................................................................. 9
3.2.4.1. Residue studies ........................................................................................................................... 9
3.2.4.2. Assessment of Consumer exposure ............................................................................................... 10
3.2.4.3. Conclusions on safety for the consumer ........................................................................................ 10
3.2.5. Safety for the user ...................................................................................................................... 10
3.2.5.1. Conclusions on safety for the user ................................................................................................ 11
3.2.6. Safety for the environment .......................................................................................................... 11
3.3. Efﬁcacy ............................................................................................................................... ....... 11
3.3.1. Poultry ............................................................................................................................... ......... 11
3.3.1.1. Conclusions on ef ﬁcacy in poultry ................................................................................................. 12
3.3.2. Pigs............................................................................................................................... ............. 12
3.3.2.1. Conclusions on the ef ﬁcacy in pigs ................................................................................................ 13
3.3.3. Ruminants ............................................................................................................................... ... 13
3.3.3.1. Conclusions on the ef ﬁcacy in ruminants ....................................................................................... 13
3.3.4. Aquatic animal species ................................................................................................................. 13
3.3.4.1. Conclusions on the ef ﬁcacy in aquatic animal species ..................................................................... 14
3.3.5. Conclusions on ef ﬁcacy ................................................................................................................ 14
3.4. Post-market monitoring ................................................................................................................ 14
4. Conclusions ............................................................................................................................... .. 14
5. Recommendation ........................................................................................................................ 14
6. Documentation provided to EFSA/Chronology ................................................................................ 14
References ............................................................................................................................... ................ 15
Abbreviations ............................................................................................................................... ............ 17Guanidinoacetic acid for all animal species
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1. Introduction
1.1. Background and Terms of Reference
Regulation (EC) No 1831/20031establishes the 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 Trostberg GmbH2for authorisation of
the product guanidinoacetic acid when used as a feed additive for all animal species (categorynutritional additives, functional group amino acids, their salts and analogues).
According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the
application to the European Food Safety Authority (EFSA) as an application under Article 4(1)(authorisation of a feed additive or new use of a feed additive). The particulars and documents in
support of the application were considered valid by EFSA as of 28 September 2020.
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 whetherthe feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion onthe safety for the target animals, consumer, user and the environment and on the ef ﬁcacy of the
product guanidinoacetic acid, when used under the proposed conditions of use (see Section 3.1.4 ).
1.2. Additional information
The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of EFSA issued
two opinions on the safety and ef ﬁcacy of guanidinoacetic acid for chickens for fattening and for pigs
(EFSA, 2009; EFSA FEEDAP Panel, 2016).
GAA is authorised (3c372) as nutritional additive for chickens for fattening, weaned piglets and pigs
for fattening.3
2. Data and methodologies
2.1. Data
The present assessment is based on data submitted by the applicant in the form of a technical
dossier4in support of the authorisation request for the use of guanidinoacetic acid as a feed additive.
The FEEDAP Panel used the data provided by the applicant together with data from other sources,
such as previous risk assessments by EFSA or other expert bodies, peer-reviewed scienti ﬁc papers,
other scienti ﬁc reports and experts ’knowledge, to deliver the present output.
The European Union Reference Laboratory (EURL) considered that the conclusions and
recommendations reached in the previous assessment regarding the methods used for the control of
guanidinoacetic acid in animal feed are valid and applicable for the current application.5
2.2. Methodologies
The approach followed by the FEEDAP Panel to assess the safety and the ef ﬁcacy of
guanidinoacetic acid is in line with the principles laid down in Regulation (EC) No 429/2008 and therelevant guidance documents: Guidance on studies concerning the safety of use of the additive forusers/workers (EFSA FEEDAP Panel, 2012), Guidance on the identity, characterisation and conditions ofuse of feed additives (EFSA FEEEDAP Panel, 2017a), Guidance on the assessment of the safety of feedadditives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of thesafety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c), Guidance on the assessment
1Regulation (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.
2Alzchem Trostberg GmbH. Dr. Albert Frank Str. 32, 83308 Trostberg, Germany.
3Commission implementing regulation (EU) 2016/1768 of 4 October 2016 concerning the authorisation of guanidinoacetic acidas a feed additive for chickens for fattening, weaned piglets and pigs for fattening and repealing Commission Regulation (EC)No 904/2009. L 270/4, OJ 5.10.2016, 3 pp.
4FEED dossier reference: FAD-2020-0050.
5The full report is available on the EURL website: https://ec.europa.eu/jrc/sites/jrcsh/ ﬁles/FinRep-FAD-2011-0043-GAA.doc_.pdfGuanidinoacetic acid for all animal species
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of the ef ﬁcacy of feed additives (EFSA FEEDAP Panel, 2018), Guidance on the assessment of the
safety of feed additives for the environment (EFSA FEEDAP Panel, 2019).
3. Assessment
The subject of this assessment is guanidinoacetic acid (GAA) when used as a nutritional feed
additive (functional group: amino acids, their salts and analogues) for all animal species. The additiveis currently authorised for its use in chickens for fattening, weaned piglets and pigs for fattening, andthe current application is for the extension of its use in feed for all animal species.
3.1. Characterisation
The manufacturing process and the characterisation of the active substance were already described
in a previous opinion (EFSA FEEDAP Panel, 2016).
3.1.1. Characterisation of the guanidinoacetic acid crystalline powder
The product is currently authorised as a powder with a minimum content of 98% GAA (on DM
basis) and a maximum of 0.5% dicyandiamide and 0.03% cyanamide. The applicant requests to keep
the same speci ﬁcations.
The analysis of ﬁve batches (ion chromatography) showed an average of 98.7% of GAA as is (range
98.3 –99.2%) and moisture ranged <0.1–0.2%. Other components analysed were glycine that averaged
2,500 mg/kg (range 1,700 –3,200 mg/kg), dicyandiamide 760 mg/kg (range 500 –1,000 mg/kg),
melamine 3.7 mg/kg (range 2.1 –4.7 mg/kg). Cyanamide was found below the limit of quanti ﬁcation
(LOQ, 7 mg/kg) in all ﬁve batches.6Ammeline, ammelide and cyanuric acid were analysed in ﬁve batches.
On average, ammeline content was 1.3 mg/kg (range 0.8 –1.9 mg/kg), ammelide was 0.14 mg/kg (range
0.06 –0.31 mg/kg) and cyanuric acid was <LOQ (0.05 mg/kg) in all batches.6
The level of cadmium, lead, mercury and arsenic was analysed in three batches of GAA, all showing
values below the respective LOQ.7
The detected amount of these impurities does not represent safety concerns.GAA is a white crystalline powder having a solubility in water of 1.5 g/L at 15 °C and of 5.5 g/L at
20°C. Bulk density measured in three batches ranged from 340 to 370 kg/m
3while tapped density
ranged from 560 to 590 kg/m3.
The dusting potential was analysed (Stauber-Heubach method) in three batches of the GAA
crystalline and it ranged from 3.8 to 7.5 g/m3.8Particle size distribution of GAA was measured in six
batches by laser diffraction and the fractions of particles with a diameter <11,<50 and <105 µm
were 40 –44%, 93 –94% and 100%, respectively.9
3.1.2. Characterisation of the formulated additive
The additive should also be placed on the market in a formulated form (Creamino®), which is
speci ﬁed to contain ≥96% GAA, ≤1% water and ~1% starch.
The speci ﬁcation was con ﬁrmed by the analysis of ﬁve batches, showing a mean of 98.0% GAA
(range 97.4 –98.7%) and 0.2% moisture (range <0.1–0.3%).10Melamine content was on average
4.4 mg/kg (range 3 –6 mg/kg).
The formulated additive is an off-white granular product. Its solubility in water is 5.53 g/L at
20/C176C.11It has a bulk density (three batches) ranging from 605 to 610 kg/m3and a tapped density
(three batches) ranging from 690 to 694 kg/m3.12
The dusting potential of the formulated additive was analysed in three batches (Stauber-Heubach)
and ranged from 5.12 to 6.15 g/m3.12The particle size distribution measured by sieving of 15 batches
6Technical dossier/Supplementary information August 2021/Annex II.2.1.3.1 updated.
7Technical dossier/Supplementary information August 2021/Annex II.2.1.3.1 updated. LOQ in mg/kg was 0.02 for cadmium
and arsenic, 0.04 for mercury and 0.32 for lead.
8Technical dossier/Section II/Annex II.2.1.5.7.
9Technical dossier/Section II/Annex II.2.1.5.5.
10Technical dossier/Supplementary information August 2021/Annex II.2.1.3.3 updated.
11Technical dossier/Section II/Annex II.2.1.5.8.
12Technical dossier/Section II/Annex II.2.1.5.6.Guanidinoacetic acid for all animal species
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showed that the fraction of particles having a diameter <63µm was on average 3% and the fraction
<100 µm was on average 4%. The mean size was on average 478 µm.13
3.1.3. Stability and homogeneity
The applicant submitted new studies on the shelf-life, stability in feedingstuffs performed with the
formulated additive; and stability in water performed with the GAA crystalline powder which
complement those already assessed in the previous opinion (EFSA FEEDAP Panel, 2016).
The shelf-life of the formulated additive was tested when stored in plastic bags at 25 °C for 5 years
(six batches) or at 40 °C for 1 year (three batches). At the end of the storage period, a loss up to 1%
was observed in half of the batches stored at 25 °C and no losses were observed in the batches stored
at 40 °C.14
The stability of the formulated additive (one batch, containing 97.6% GAA) at different
supplementation levels (600 and 1,200 mg/kg) in two complete pelleted feeds (starter and grower,
based on maize and soybean meal) for chickens for fattening was studied.15After mixing, the feeds
were conditioned and pelleted (at 75 °C for the starter and 72 °C for the grower feed). Feed processing
caused no losses of GAA. The samples were packed in paper bags and stored either at 25 °C and 60%
RH for 48 months or at 40 °C and 75% RH for 6 months. As regards the starter feed, it showed losses
of GAA ranging from 0% to 3% after storage at 25 °C and a loss of 3% after storage at 40 °C. The
grower feed showed no loss of GAA after storage at 25 °C, and losses were between 1% and 3% when
stored at 40 °C.
The stability of one batch of the formulated additive was tested in two mash feeds (starter and
grower) for chickens for fattening consisting of maize, soybean meal, DDGS and maize gluten meal
(containing ~2,600 mg/kg choline chloride), when supplemented at 600, 1,200 and 1,800 mg/kg
feed.16The samples were packed in paper bags with plastic liners and stored either at 25 °C for
12 months or at 40 °C for 6 months. As regards the starter feed, losses ranged from 0 to 5% when
stored at 25 °C and from 1% to 8% when stored at 40 °C. The grower feed showed losses ranging
from 2% to 10% when stored at 25 °C and from 0% to 11% when stored at 40 °C.
The stability of GAA crystalline powder (three batches) was tested in water at different pH (4, 7
and 9 buffered solutions and in not buffered water) and concentrations (200 and 600 mg/L, two
replicates per concentration) when stored at 25 °C for 4 days.17The solutions were tightly capped
during storage. No losses were observed at buffered solutions of pH 4 and 7 or in the non-bufferedwater. A loss of about 1% was observed in the buffered solution of pH 9 at the end of the storageperiod.
The homogeneous distribution of the additive in feed was studied in starter and grower feeds for
chickens for fattening, in mash and in pelleted form when supplemented at 600 or 1,200 mg offormulated additive/kg.
18Ten subsamples of each feed were analysed, and the coef ﬁcient of variation
ranged from 8% to 12% in mash feeds and from 2% to 4% in pelleted feeds without substantial
differences attributable to the supplementation level.
3.1.4. Conditions of use
GAA is intended to be used in feed (directly or via premixtures) for all animal species at a minimum
content of 600 mg GAA/kg complete feed and a maximum content of 1,200 mg/kg complete feed.19
GAA is also intended to be used in water for drinking for poultry and pigs,20at a minimum content of
200 mg/L and a maximum content of 600 mg/L.
3.2. Safety
To support the safety of the additive, the applicant performed an extensive literature search in
three databases (CAB abstracts, PubMed and Veterinary Science Database) including publications from
13Technical dossier/Section II/Annex II.2.1.5.1.
14Technical dossier/Section II/Annex II.2.4.1.3.
15Technical dossier/Section II/Annex II.2.4.1.7.
16Technical dossier/Section II/Annexes II.2.4.1.8 and II.2.4.1.9.
17Technical dossier/Section II/Annex II.2.4.1.11.
18Technical dossier/Section II/Annex II.2.4.1.6.
19Standardised feed with 88% dry matter.
20Technical dossier/Supplementary information August 2021/0_Creamino_cover_letter.Guanidinoacetic acid for all animal species
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1990 onwards without language restriction.21The search strategy is described in the dossier and
included terms related to the active substance and the safety and ef ﬁcacy for target animals and
safety for humans. Clear and logical inclusion and exclusion criteria were established.
3.2.1. Absorption, distribution, metabolism and excretion
The metabolic fate of GAA was described in previous opinions (EFSA, 2009; EFSA FEEDAP Panel,
2016). In summary, GAA is an endogenous compound of the biosynthetic pathway of creatine,common to all vertebrates. It is synthesised from the amino acids glycine and arginine, mainly in thekidney and pancreas. This ﬁrst step was found to be rate-limiting as the enzyme L-arginine-glycine
amidinotransferase (AGAT) is subject to feedback inhibition by creatine. The second step occurs in theliver where the enzyme GAA N-methyltransferase (GAMT) catalyses the transfer of a methyl groupfrom S-adenosylmethionine (SAM) to GAA to form creatine and S-adenosylhomocysteine (SAH). Noreverse transformation of creatine to GAA occurs. Creatine is released from the liver into the circulation
where it can be taken up, via a speci ﬁc transporter, by various tissues. Creatine, besides its energy-
related functions, undergoes an irreversible non-enzymatic conversion to creatinine which is excretedin the urine. Daily turnover of creatine must be replaced from dietary animal protein or de novosynthesis from GAA. SAH formed during this process is subsequently hydrolysed, thus generatinghomocysteine.
Some of the scienti ﬁc papers reviewed from the extensive literature review provided new
information on ADME of guanidinoacetic acid and are described below.
Tossenberger et al. (2016) showed in colostomised chickens that the apparent faecal digestibility of
GAA was 99% and 98% for GAA levels of 600 and 6,000 mg/kg complete feed, respectively (excreted at
very low levels in the faeces). The major excretion occurred in the urine principally as unchanged GAAfollowed by creatine and creatinine. Considering the renal excretion of GAA and creatine/creatinine, thetrue availability of GAA was estimated to be 76% at 600 mg/kg and 46% at 6,000 mg/kg feed.
In ruminants (cattle), about 50% of the GAA infused in the rumen (infusion up to 20 g/day) is
degraded (Speer et al., 2020). The other half reaches the abomasum and further the intestinal tract,resulting in increased plasmatic creatine and derived metabolites (Ardalan et al., 2015), the same as inother species, e.g. the chicken (see above) or humans (Edison et al., 2013). Li et al. (2020) observed
that the dietary supplementation of Angus bulls with up to 900 mg GAA/kg DM for 104 days resulted
in increased cellobiase, pectinase and protease enzymatic activities in rumen, and in propionateproduction and microbial protein synthesis (derived from lower ammonia-N) in the rumen. Theseresults suggested that dietary GAA may be used by rumen microbes to support their growth andmodulating the composition of the microbiota. Ardalan et al. (2020) showed that abomasal infusion ofGAA (10, 20, 30 and 40 g /day,
22with or without supplemental methionine) in heifers resulted in
increases of plasma creatine and the requirement for methyl donors (as evidenced by the results atthe two highest GAA infusion levels without concomitant infusion of methionine).
3.2.2. Toxicological studies
Repeated dose oral toxicity studies were already evaluated in a previous opinion (EFSA, 2009)
where it was concluded that ‘The effects reported in the 28- and 90-day studies generally re ﬂect
physiological responses to high exposures to a metabolic intermediate and do not identify any novel or
unexpected toxicity ’. No new evidence that would change the conclusions previously reached was
provided by the applicant.
The genotoxicity potential of the additive was previously evaluated (EFSA, 2009; EFSA FEEDAP
Panel, 2016) based on the assessment of an Ames test, an in vitro mammalian cell gene mutation test,and an in vitro chromosomal aberration assay. Overall, the FEEDAP Panel concluded that there was noevidence of mutagenic activity, and that the chromosomal aberration test did not show a potential forgenotoxicity of the additive under assessment.
To evaluate the potential of GAA (purity 99.2%) to induce chromosome damage (aneugenicity and
clastogenicity), an in vitro micronucleus test was performed in whole blood human lymphocytes
according to OECD Test Guideline (TG) 487 (2016) and following good laboratory practice (GLP).
23
21Technical dossier/Section III/Subsection 3.1.1.
22The estimated amount of GAA in feed to reach 10 g GAA/day in the abomasum (considering a loss of 50% GAA due to
ruminal degradation) is similar to 2,500 mg GAA/kg DM feed.
23Technical dossier/Supplementary information August 2021/Annex III.3.2.2.2.Guanidinoacetic acid for all animal species
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Based on the results of a preliminary cytotoxicity test, the compound was tested up to 10 mM applying
a short treatment (4 +40 h of recovery) in the absence and presence of metabolic activation and a
continuous treatment (44 +0 h of recovery) without metabolic activation. Exposure started 48 h after
culture initiation and the cells were cultured for a total time of 92 h. The highest concentration testedcorresponded to the top dose recommended by OECD TG 487. Appropriate positive and negativecontrol chemicals were used, and the results obtained con ﬁrmed that the experimental system was
sensitive and valid. Cytotoxicity up to 35% relative to vehicle control was observed at 10 mM aftershort treatment with metabolic activation. The analysis of micronuclei was performed in binucleated
cells blocked by 6 µg/mL cytochalasin B. No signi ﬁcant increase in the frequency of micronucleated
cells was induced by GAA at any tested concentration. Based on these results, the Panel concludedthat GAA did not induce structural and numerical chromosomal damage under the experimentalconditions applied in this study.
3.2.3. Safety for the target species
The FEEDAP Panel, on the basis of tolerance studies in chickens for fattening and weaned piglets,
concluded in its previous opinion that a concentration of 1,200 mg GAA/kg complete feed could beconsidered safe for chickens for fattening, weaned piglets and pigs for fattening, given that anadequate dietary supply of methyl donors is provided (EFSA FEEDAP Panel, 2016). In the absence ofadequate data, the FEEDAP Panel could not conclude on the safety of GAA for breeder hens androosters.
To support the safety of the additive for all animal species, the applicant provided a tolerance study
in laying hens and conducted an extensive literature search (described at the start of Section 3.2).
From a total of 885 references retrieved, 73 were considered relevant after screening title andabstract, and subsequently reduced to 20 after full-text revision.
24From these 20, 19 references were
not further considered because (i) they were studies already considered in the previous opinion (EFSAFEEDAP Panel 2016), (ii) the studies did not include relevant endpoints for the safety assessment or(iii) there were some de ﬁciencies in the study design or execution. Therefore, only one publication was
considered relevant by the Panel.
The literature reviewed did not provide new information on the safety of the additive for chickens
for fattening or pigs.
3.2.3.1. Laying hens
The applicant submitted a new tolerance study in laying hens.
25A total of 390 Lohmann Brown classic
laying hens of 25 weeks of age were randomly distributed in 30 ﬂoor pens and the pens allocated to one
of the ﬁve dietary treatments (six replicates/treatment). The experimental diet, based on maize and
soybean meal and containing <20 mg creatine/kg, 4 g methionine/kg, 1,200 mg choline/kg, 0.9 mg folic
acid/kg and 17 µg vitamin B 12/kg, was either not supplemented (control) or supplemented with the
formulated additive at 1,200 (1 9maximum use level), 3,600 (3 9), 6,000 (5 9) or 12,000 (10 9) mg GAA/
kg feed (con ﬁrmed by analysis) and fed for 64 days. Body weight of the hens was determined in 4-week
period (pen basis). Feed intake was determined weekly per pen. Pen laying rate and egg mass wererecorded daily. Individual egg weight and egg quality parameters (egg yolk colour, weight and shell indexof dried shells) were determined on days 28 and 56. Two eggs per pen (12/treatment) were randomlycollected on day 57, freeze-dried (whole egg) and analysed for GAA, creatine, creatinine andhomocysteine contents. Two hens per pen (12/treatment) were killed on day 62 and subjected tonecropsy, and two other hens per pen were sampled on day 64 for blood analysis (for haematology
26and
clinical chemistry,27including GAA, creatine, creatinine and homocysteine). Data were statistically
analysed by ANOVA using the pen as a statistical unit for performance data and the individual animal (oregg) for blood parameters, necropsy data and metabolites in eggs. Differences between groups weretested with Tukey test and the signi ﬁcance was set at p <0.05.
24Technical dossier/Section III.3.1.1.
25Technical dossier/Supplementary information August 2021/Annex III.3.2.1.2.3.
26Haematological analyses consisted of determination of erythrocytes, haemoglobin, haematocrit, mean cell volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), heterophils, monocytes,
lymphocytes, thrombocytes, T-cell and B-cell counts.
27Clinical chemistry consisted of plasmatic determination of aspartate aminotransferase (AST), gamma-glutamyl transferase
(GGT), alkaline phosphatase (AP), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), total
protein, albumin, cholesterol, triglycerides, bile acids, urea, uric acid, sodium, potassium, chloride, calcium, magnesium and
phosphorous.Guanidinoacetic acid for all animal species
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Mortality was low (two birds in the 5 9group and one in the 10 9group) and not treatment related.
Hens ’body weight at the end of the study was signi ﬁcantly lower in the groups treated with 6,000 and
12,000 mg GAA/kg compared to the control (1,858 and 1,882 g vs 2,011 g, respectively). Daily feedintake was signi ﬁcantly reduced in all treatment groups (121, 119, 120 and 113 g/d vs 130 g/d in the
control group) and there was no improvement in feed to egg mass ratio (2.12 in the control group).Laying rate was signi ﬁcantly reduced in the group treated with 12,000 mg GAA/kg feed (87% vs 98%
in the control group). Average egg weight was signi ﬁcantly reduced in the groups treated with 1,200
and 12,000 mg GAA/kg feed (60.2 and 59.7 g vs 62.4 g in control group) and daily egg mass was
signi ﬁcantly reduced in groups treated with 1,200, 6,000 and 12,000 mg GAA/kg feed (57.2, 57.2 and
51.8 vs 61.0 g/day in control group). No differences were reported in yolk or eggshell weights.Regarding egg yolk colour, there were differences between treatments but not dose related. Nodifferences in haematological or clinical chemistry parameters were observed (except albumin,cholesterol and triglycerides that were signi ﬁcantly decreased and glucose that was signi ﬁcantly
increased in the 10 9group). Creatinine in plasma was below the LOQ in all samples. Plasmatic GAA
and creatine increased in a dose-related manner. Analytical values were 10, 30, 59 and 175 µmol GAA/L,
for the treatments with 1,200, 3,600, 6,000 and 12,000mg/kg, respectively, vs. 0.4 µmol/L in control
group; and 2.2, 3.2, 4.0 and 5.5 mg creatine/dL, respectively, vs. 1.6 mg/dL in control group.
Homocysteine in plasma was signi ﬁcantly increased in the groups receiving 3,600, 6,000 and 12,000 mg
GAA/kg feed (analytical values 23, 25 and 32 µmol homocysteine/L, respectively, vs. 13 µmol/L in control
group). No relevant necropsy or histopathological ﬁndings were observed.
The results of the study showed statistically signi ﬁcant negative effects of the GAA supplementation
from the maximum proposed use level of 1,200 mg GAA/kg feed on daily feed intake, average eggweight and daily egg mass.
3.2.3.2. Fish
Nile tilapia ( Oreochromis niloticus ) were fed diets containing GAA up to 1.5 9the maximum use
level for 60 days (Aziza et al., 2020). Growth performance, biochemical parameters and antioxidantcapacity were measured. Although the diet contained 20% ﬁshmeal, the supplementation of the diet
with 600, 1,200 and 1,800 mg GAA/kg feed resulted in a dose-related improvement of the ﬁnal body
weight, reaching signi ﬁcance at the two highest levels. Speci ﬁc growth ratio of ﬁsh treated at
1,800 mg/kg was not signi ﬁcantly different compared to the control. Body composition was unaffected
with the exemption of lipid % that decreased at the highest GAA level. Serum creatine kinase was
signi ﬁcantly increased in all GAA-treated groups, creatinine and cholesterol in the 1,200 and 1,800 mg
GAA/kg diet groups and triglycerides only in the group receiving 1,800 mg GAA/kg feed in comparisonwith the control diet. There were no differences between diets regarding the following bloodparameters: total protein, globulin, albumin or albumin:globulin. However, the short duration of thestudy, the low range of experimental doses tested and the inadequacy of the endpoints assessedprevented to reach a conclusion on the safety of GAA for tilapia.
3.2.3.3. Conclusions on safety for the target species
No new data were submitted that would lead the Panel to modify its previous conclusions that GAA
at 1,200 mg/kg complete feed is safe for chickens for fattening, piglets and pigs for fattening. Thisconcentration in complete feed would correspond to maximum concentrations in water for drinking of600 mg GAA/L for chickens for fattening, piglets and pigs for fattening. The conclusions on chickensfor fattening can be extended to chickens reared for breeding/laying.
The new tolerance study submitted in laying hens indicates that 1,200 mg/kg is not tolerated.
Therefore, the Panel is not in a position to conclude on a safe level of GAA in laying/reproductive birds.
In the absence of data on ruminants and salmonids, the FEEDAP Panel cannot conclude on the
safety of GAA for all animal species.
3.2.4. Safety for the consumer
3.2.4.1. Residue studies
The previous opinions assessed the deposition in tissues of GAA, its metabolites creatine and
creatinine and homocysteine based on data from four studies in chickens for fattening (EFSA, 2009) oron data from nine studies on chickens for fattening and one in pigs (EFSA FEEDAP Panel, 2016). The
FEEDAP Panel concluded that there is no concern on consumer safety resulting from the use of GAA in
feed for chickens for fattening and piglets/pigs for fattening at 1,200 mg GAA/kg feed.Guanidinoacetic acid for all animal species
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No new data were submitted that would modify the previous observations on concentrations of
GAA, creatine and creatinine in chicken muscle and liver (EFSA FEEDAP Panel, 2016).
As the current application includes the extension of use to all animal species (including ruminants,
laying hens and ﬁsh), the potential exposure of the consumer through tissues and products from these
animals should be estimated. The applicant provided data on deposition in eggs of laying hens andquails, and in ﬁsh. However, the studies in ﬁsh were not considered adequate as they tested levels far
above the maximum proposed use level and did not measure the levels of homocysteine.
No data on deposition of GAA or its metabolites in tissues or milk from ruminants were submitted.
The applicant proposed to assess exposure via milk by comparing the data on residues in milk from
sows fed diets supplemented with GAA (see Section 3.3.2 ) with that of milk obtained from animals of
other species fed diets not supplemented with GAA.
28The FEEDAP Panel considers that the data from
sows cannot be used as surrogate for cow ’s milk due to the differences in feeding conditions and GAA
metabolism between cattle and pigs.
In the tolerance study in laying hens (Section 3.2.3.1 ),25GAA, creatine and creatinine levels were
analysed at the end of the 56-day experimental period.
Supplementing GAA up to the maximum use level did not result in a signi ﬁcant increase in GAA,
creatinine or homocysteine deposition in eggs (Table 1). However, creatine deposition was signi ﬁcantly
increased with GAA supplementation.
Murakami et al. (2014) analysed the contents of GAA, creatine and creatinine in eggs of quails that
had been fed with GAA at 0 and 1,200 mg GAA/kg complete feed for 4 weeks. Supplementation withGAA caused marginal increases in GAA (0 vs. 1.5 µg/egg) and creatine (19.6 vs. 24.1 µg/egg)
concentrations in eggs in comparison with the not supplemented control.
3.2.4.2. Assessment of Consumer exposure
In the previous opinion, the Panel concluded that no additional exposure of the consumer to GAA,
creatine, creatinine and homocysteine was expected from the use of GAA in feed for chickens forfattening and piglets up to 1,200 mg/kg feed (EFSA FEEDAP Panel, 2016).
A similar conclusion can be reached based on the results observed in deposition of GAA, creatinine
and homocysteine in eggs from laying hens fed GAA at 1,200 mg/kg complete feed. Although creatineconcentration in eggs was signi ﬁcantly increased with regard to the control, these levels would be in
the range (3.3 –5.9 mg/kg egg) reported in the scienti ﬁc literature (Comert and Gokmen, 2020; Reicher
et al., 2020).
29The marginal increases in deposition observed in quail eggs are not considered to
appreciably increase the exposure of consumers.
In the absence of data in milk and tissues from ﬁsh, no conclusion can be reached on the exposure
of consumers to GAA or its metabolites from the use of GAA in ruminants and ﬁsh.
3.2.4.3. Conclusions on safety for the consumer
The FEEDAP Panel concludes that there is no concern for consumer safety resulting from the use of
GAA in feed for chickens for fattening, piglets/pigs for fattening and laying hens under the proposedconditions of use (1,200 mg/kg feed). Due to the limited data available, the FEEDAP Panel cannot
conclude on the safety of the additive for the consumer when included in feeds for ruminants and ﬁsh.Table 1: Deposition of GAA and its metabolites in eggs of hens fed with GAA at 1,200 mg/kg
complete feed. Values in mg/kg egg, considering 25% DM in egg
GAA, mg/kg feed 0 1,200
GAA 0.20 0.28
Creatine 4.00a5.50b
Creatinine 0.43 0.5
Homocysteine 0.90 1.08
Different superscripts in a row are indicative of signi ﬁcant differences (p <0.05).
28Technical dossier/Supplementary information August 2021/Annex III.3.2.1.2.1.
29In the study by Reicher et al 2020, the creatine values were expressed in mg/egg freeze dried and they had to be converted
to mg/kg egg. Default values used to transform units reported into mg/kg fresh egg: moisture in whole egg is 75%, in egg
white is 88% and in egg yolk is 51%; mass of whole edible egg consists of 66% white egg and 34% egg yolk. Molecular
weight of creatine = 131.13 g/mol.Guanidinoacetic acid for all animal species
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3.2.5. Safety for the user
The highest dusting potential measured for GAA (crystalline form) was 7.5 g/m3and its particle size
distribution showed that practically all particles had a diameter <100 µm. The respective values for
the formulated additive were 6.2 g/m3and 4% of particles <100 µm diameter. Based on these data,
the FEEDAP Panel considered that the exposure of users through inhalation is likely.
In a valid acute inhalation toxicity study performed according to OECD Test Guideline 403 and
compliant with good laboratory practice (GLP), the active substance GAA showed an inhalation medianlethal dose >5.1 mg/L air.
30
The studies on skin and eye irritancy and on skin sensitisation submitted were already assessed in
the former opinion (EFSA, 2009). GAA was considered not irritant to eyes and skin and not a dermalsensitiser. No new data have been submitted that would lead to reconsider the conclusions previouslyreached.
3.2.5.1. Conclusions on safety for the user
Based on the data available, the FEEDAP Panel concludes that GAA is not toxic by inhalation, it is
not an irritant to skin and eyes, and it is not a dermal sensitiser.
3.2.6. Safety for the environment
The safety for the environment was assessed in a previous opinion (EFSA, 2009) and the extension
of use to new species is not expected to modify the outcome of the assessment. GAA is a physiologicalmolecule that occurs naturally in animals, and its use as a feed additive would not lead to excretion ofmolecules which are not normally present in the excreta of the target animals. The FEEDAPPanel concludes that the use of GAA as a feed additive is not expected to pose a risk to theenvironment.
3.3. Ef ﬁcacy
The applicant requested the authorisation of GAA as a nutritional additive, functional group amino
acids, their salts and analogues. In the view of the FEEDAP Panel, this functional group 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. Although resulting from the aminoacid metabolism, GAA is exclusively converted to creatine and cannot be converted back to an aminoacid. Therefore, the Panel considers that to support the ef ﬁcacy of this product as a novel nutritional
additive, relevant evidence of its ef ﬁcacy should be provided at least in poultry, pigs, ruminants and
ﬁsh, covering both growing and reproductive animals.
The applicant conducted an extensive literature search (described under Section 3.2). From a total
of 885 references retrieved, the applicant selected 53 (2 of them EFSA opinions) as relevant after
screening of title and abstract and the review of the full text. Eleven additional scienti ﬁc papers
originating from other sources (unspeci ﬁed) were also included in the list of relevant references.
31
3.3.1. Poultry
The FEEDAP Panel concluded in a previous opinion (EFSA FEEDAP Panel, 2016) that GAA is able to
improve the performance in chickens for fattening at the minimum concentration of 600 mg/kg
complete feed. In the absence of adequate data, the FEEDAP Panel could not conclude on the ef ﬁcacy
of GAA in breeder hens and roosters.
From the 48 publications provided by the applicant, nine were considered relevant. The rest were
not further considered because there were some de ﬁciencies in the study design or execution (e.g.
short duration of the treatment, or the effect was studied in conditions of hypoxia, or there were noreplicates, or the control group did not perform as expected, or doses tested were outside therecommended range).
Several studies in chickens for fattening (e.g. Metwally et al., 2015; Malins et al., 2017; Yapontsev
et al., 2017; and C /C19ordova-Noboa et al., 2018) support the previous conclusion on the lowest effective
dose of GAA (600 mg/kg complete feed) (EFSA FEEDAP Panel, 2016).
30Technical dossier/Section III/Annex III.3.2.2.1.
31Technical dossier/Section IV.4.3.Guanidinoacetic acid for all animal species
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Ringel et al. (2007) studied the effect of supplementing GAA at 400 or 800 mg GAA/kg feed for
20 weeks on the performance of turkeys for fattening. The supplementation with a concentration of800 mg GAA/kg feed signi ﬁcantly reduced feed intake and improved feed to gain ratio (2.66 vs. 2.72),
with no improvements in the body weight gain. Carcass weight and yield were unaffected bytreatments. Breast weight percentage was higher in turkeys fed 400 or 800 mg GAA/kg and leg weightpercentage was lower at 800 mg GAA/kg.
Morshedy et al. (2019
) studied the effect of supplementing GAA at 500 mg/kg feed with or without
supplemental methionine on the performance of ducks for fattening and meat parameters. GAA
supplemented at 500 mg/kg feed for 42 days improved ducks ’performance parameters (body weight
(BW), body weight gain (BWG), feed to gain ratio and protein ef ﬁciency ratio) and carcass traits.
Kuhi Darmani et al. (2019) studied the effect of supplementing GAA at 600 or 1,200 mg/kg feed
and L-arginine for 42 days on performance, blood metabolites and carcass characteristics of Japanesequails. Supplementing a diet covering arginine requirements for quails for fattening with 600 or1,200 mg GAA/kg feed had no effect on performance parameters (feed intake (FI), BWG and feed togain ratio) or carcass traits.
Khakran et al. (2017) studied the effect of supplementing GAA ranging from 570 to 1,710 mg/kg
feed for 84 days on productive performance, some blood biochemical parameters and reproductive
hormones of laying hens. No signi ﬁcant effects were seen in the feed intake, laying rate, egg mass
and feed to egg mass ratio. Supplementation at 1,710 mg GAA/kg feed caused a reduction in eggweight.
Tapeh et al. (2017) studied the effect of supplementing GAA diet supplementation on semen quality
and fertility of breeder roosters for chickens for fattening when added from 600 to 1,800 mg/kg feedfor 26 weeks. Supplementing complete feed with 1,200 mg GAA/kg showed improvements in thereproductive capacity of roosters. However, as concurrent negative and positive effects were reported
at the 600 mg/kg (lower sperm concentration, higher fertility), a clear conclusion at the recommended
levels cannot be drawn.
3.3.1.1. Conclusions on ef ﬁcacy in poultry
Based on the new studies provided, the Panel con ﬁrms its previous conclusion that 600 mg GAA/kg
feed is ef ﬁcacious in improving the performance of chickens for fattening. Similar results were
observed in turkeys for fattening (800 mg/kg) and ducks (500 mg/kg), but not in quail. Consideringthe overall data, the Panel concludes that GAA at a minimum level of 600 mg/kg complete feed has
the potential to improve the zootechnical performance of all growing avian species. No conclusion can
be reached on reproductive avian species.
3.3.2. Pigs
As regards growing pigs, one valid study on the ef ﬁcacy of GAA in weaned piglets submitted in a
previous opinion showed that 1,200 mg/kg complete feed improved growth ( ﬁnal BW and average
daily gain (ADG)) (EFSA FEEDAP Panel, 2016). The applicant submitted eight new studies. Six of themwere not considered further due to the short duration of the trials.
He et al. (2018) studied the effects of dietary GAA supplementation at 300, 900 and 1,200 mg/kg
feed for 14 weeks on growth performance, creatine and energy metabolism, and carcasscharacteristics in grower –ﬁnisher pigs. GAA supplementation signi ﬁcantly improved gain to feed ratio at
300, 900 and 1,200 mg GAA/kg feed; and the hot carcass weight at 300 and 1,200 mg/kg. GAAtreatments did not signi ﬁcantly affect serum (GAA, creatine, creatinine and creatine kinase [CK]) or
tissue concentrations (GAA, creatine in muscle, liver or kidney, and CK in muscle) of energy-related
metabolites, except serum ATP which was signi ﬁcantly increased in a dose-dependent manner with the
GAA feed content.
Jayaraman et al. (2018
) studied the effect of the dietary supplementation of 800 or 1,200 mg GAA/
kg feed to pig diets for 150 days on performance, carcass characteristics and meat quality.Performance parameters ( ﬁnal BW, ADG and feed to gain ratio) were signi ﬁcantly improved with GAA
supplementation at any level. Some carcass parameters (% of lean meat, back fat) were signi ﬁcantly
improved at GAA supplementation of 1,200 mg/kg.
Considering pigs for reproduction, the applicant provided a new study in pregnant sows and their
progeny of 42-day duration (from gestation day 100 to weaning –day 28 post-farrowing).
28The basal
diets (gestation and lactation) were either not supplemented (control) or supplemented with 1,000,5,000 or 10,000 mg GAA/kg feed (con ﬁrmed by analysis). No relevant signi ﬁcant differences betweenGuanidinoacetic acid for all animal species
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treatments were observed in sows ’performance parameters (percentage of BW loss in lactation,
percentage of back fat loss in lactation, feed consumption (FI)); or in piglets performance parameters(ﬁnal BW, litter homogeneity, ADG, pre-weaning mortality).
Mendonc /C223a et al. (2019) studied the effect of the dietary supplementation with 1,000 mg GAA/kg
feed for sows and their progenies (one production cycle) in nursery pigs ’performance and blood
parameters. Data on reproductive performance of sows (e.g. reproductive data, total born, weanedpigs/sow) were not provided. Dietary supplementation with 1,000 mg GAA/kg of sows and theirprogenies did not affect performance parameters (average daily feed intake, ADG and feed to gain)
and creatinine and creatine kinase serum concentrations of piglets at nursery.
3.3.2.1. Conclusions on the ef ﬁcacy in pigs
Considering the three studies in which positive effects on performance of growing pigs were
observed at levels ranging 300 –1,200 mg GAA/kg feed, the Panel concludes that the dietary
supplementation of GAA under the proposed conditions of use has the potential to improve thezootechnical performance in growing Suidae . In the absence of adequate evidence, no conclusions can
be reached regarding the ef ﬁcacy in reproductive pigs.
3.3.3. Ruminants
As regards growing ruminants, Li et al. (2020) studied the effect of the dietary supplementation
with GAA from 300 to 900 mg/kg DM on the performance, nutrient digestion, rumen fermentation andblood metabolites in Angus bulls of 1 year of age. Signi ﬁcant improvements in ADG and ﬁnal BW
were seen in all treated groups and DM intake and feed to gain signi ﬁcantly improved at 600 and
900 mg/kg DM.
Chao et al. (2019) studied the effect of the dietary supplementation with GAA from 400 to
1,200 mg/kg feed for 85 days on growth performance, slaughter performance, fat deposition andnutritional components in muscle of Chinese Tan rams. The supplementation of weaned Tan rams ’diet
with 800 mg GAA/kg feed improved performance ( ﬁnal BW, ADG, feed to gain ratio), carcass
characteristics (carcass yield, carcass ’lean meat percentage, tail fat, subcutaneous fat thickness and
marbling score) and nutritional composition of meat (glycogen, protein and intramuscular fat content)
in comparison with the control diet.
3.3.3.1. Conclusions on the ef ﬁcacy in ruminants
The dietary supplementation with a range of 300 –900 mg GAA/kg DM feed is considered ef ﬁcacious
for improving the zootechnical performance of growing ruminants. In the absence of data, noconclusions can be reached for young (milk/milk replacer fed) ruminants and for reproductiveruminants.
3.3.4. Aquatic animal species
The applicant submitted scienti ﬁc papers in relation to the ef ﬁcacy of the additive in Nile tilapia
(Oreochromis niloticus ), Jian carps ( Cyprinus carpio var. Jian) and North American bullfrog ( Lithobates
catesbeianus ).
Aziza et al. (2019) assessed the effect of supplementing 600, 1,200 and 1,800 mg GAA/kg feed for
60 days on Nile tilapia ’s(Oreochromis niloticus ) growth performance. Body weight gain improved with
supplementation levels above 600 mg GAA/kg feed. Final body weight, body weight gain and protein
efﬁciency ratio improved at 1,200 and 1,800 mg GAA/kg feed. Speci ﬁc growth rate was increased at
1,200 mg GAA/kg feed. Dietary supplementation of Nile tilapia ’s feed with 1,200 and 1,800 mg GAA/kg
feed improved performance parameters.
Fu et al. (2015) studied the effect of supplementing 250, 500 and 1,000 mg GAA/kg feed for
42 days on growth performance, and body composition in Jian carps ( Cyprinus carpio var. Jian). The
supplementation of 250 and 500 mg of GAA/kg feed improved feed to gain ratio. No other differenceswere seen in performance parameters or carcass composition, except for an increased viscera relative
weight in the groups treated with GAA.
Zeng et al. (2018) studied the effect of supplementing 200, 400, 600 and 800 mg GAA/kg feed for
53 days in all-plant protein diets on growth and muscle energy metabolism of North American bullfrog(Lithobates catesbeianus ). The supplementation of 200 mg GAA/kg feed improved protein ef ﬁciency
ratio in comparison with the control diet. The supplementation of 400 mg of GAA/kg feed improvedﬁnal BW, weight gain, speci ﬁc growth rate, feed to gain ratio and protein ef ﬁciency ratio in comparisonGuanidinoacetic acid for all animal species
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with the control diet. As regards carcass indexes, in comparison with the control, all treatments
decreased the hepatosomatic index, and 600 and 800 mg GAA/kg increased hind leg index.
3.3.4.1. Conclusions on the ef ﬁcacy in aquatic animal species
The dietary supplementation with GAA at the proposed use range (600 –1,200 mg GAA/kg) has the
potential to improve zootechnical performance of growing ﬁsh other than salmonids. Even lower GAA
levels seem to be ef ﬁcacious in carp and bullfrog. The FEEDAP Panel cannot conclude on the ef ﬁcacy
of the additive under assessment for salmonids or for crustaceans.
3.3.5. Conclusions on ef ﬁcacy
The use of the additive under assessment in animal nutrition at the proposed conditions of use has
the potential to be ef ﬁcacious in all growing avian, Suidae, ruminant (except for pre-ruminants)
species; in growing ﬁnﬁsh other than salmonids and in frog. It is not possible to conclude on the
efﬁcacy of the additive in other species, and in reproductive animals.
3.4. Post-market monitoring
The FEEDAP Panel considers that there is no need for speci ﬁc requirements for a post-market
monitoring plan other than those established in the Feed Hygiene Regulation32and Good
Manufacturing Practice.
4. Conclusions
The FEEDAP Panel concludes that GAA at 1,200 mg/kg complete feed is safe for chickens for
fattening, chickens reared for breeding/laying, piglets and pigs for fattening. This level in complete feedwould correspond to maximum concentrations in water of 600 mg GAA/L. The Panel is not in a position
to conclude on a safe level of GAA in laying/reproductive birds. In the absence of data on ruminants and
salmonids, the FEEDAP Panel cannot conclude on the safety of GAA for all animal species.
There is no concern on consumer safety resulting from the use of GAA in feed for poultry and pigs
at the proposed conditions of use. The limited data do not allow to conclude on the safety for theconsumer when the additive is used in feed for ruminants or ﬁsh.
GAA is not toxic by inhalation, it is not an irritant to skin and eyes and it is not a dermal sensitiser.The FEEDAP Panel concludes that the use of GAA as feed additive is not expected to pose a risk to
the environment.
The use of the additive under assessment in animal nutrition at the proposed conditions of use has
the potential to be ef ﬁcacious in all growing avian, Suidae, ruminant (except for pre-ruminants)
species; in growing ﬁnﬁsh other than salmonids and in frog. It is not possible to conclude on the
efﬁcacy of the additive in other species, and in reproductive animals.
5. Recommendation
The maximum safe levels of GAA in feed for chickens for fattening, piglets and pigs for fattening
are derived under the assumption that the feed contains suf ﬁcient 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 the ‘other provisions ’of the authorisation.
6. Documentation provided to EFSA/Chronology
Date Event
30/06/2020 Dossier received by EFSA. Guanidinoacetic acid for all animal species. Alzchem Trostberg
GmbH.
17/07/2020 Reception mandate from the European Commission
28/09/2020 Application validated by EFSA –Start of the scienti ﬁc assessment
32Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for
feed hygiene. OJ L 35, 8.2.2005, p. 1.Guanidinoacetic acid for all animal species
www.efsa.europa.eu/efsajournal 14 EFSA Journal 2022;20(5):7269
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Date Event
30/11/2020 Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation
(EC) No 1831/2003 –Scienti ﬁc assessment suspended. Issues: Characterisation of the additive,
conditions of use, safety for the consumer .
05/01/2021 Comments received from Member States
24/08/2021 Reception of supplementary information from the applicant - Scienti ﬁc assessment re-started
24/03/2022 Opinion adopted by the FEEDAP Panel. End of the Scienti ﬁc assessment
28/09/2022 The FEEDAP Panel withdrew the opinion adopted on 24/03/2022. The amended opinion was
adopted by the FEEDAP Panel.
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Abbreviations
ADFI average daily feed intake
ADG average daily gain
ADI acceptable daily intakeAFC EFSA Scienti ﬁc Panel on Food Additives, Flavourings, Processing Aids and Materials in
Contact with Food
ANS EFSA Scienti ﬁc Panel on Additives and Nutrient Sources added to Food
BW body weightCAS Chemical Abstracts ServiceCFU colony-forming unit
CV coef ﬁcient of variation
DM dry matterEINECS European Inventory of Existing Chemical SubstancesEURL European Union Reference LaboratoryFCR feed conversion ratioFEEDAP EFSA Scienti ﬁc Panel on Additives and Products or Substances used in Animal Feed
IUPAC International Union of Pure and Applied ChemistryLOD limit of detection
LOQ limit of quanti ﬁcation
MRL maximum residue limitNOAEL no observed adverse effect levelOECD Organisation for Economic Co-operation and DevelopmentGuanidinoacetic acid for all animal species
www.efsa.europa.eu/efsajournal 17 EFSA Journal 2022;20(5):7269
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