The EFSA Journal (2005) 195, 1-10

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1 The EFSA Journal (2005) 195, 1-10 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety and the bioavailability of product L-Histidine monohydrochloride monohydrate for salmonids (Question N o EFSA-Q ) Adopted on 2 March 2005 SUMMARY The product contains 74 % of the essential amino acid L-Histidine and is to be used in supplementing salmon feed. It is an essential amino acid also for fish, and has been shown to prevent cataracts under practical farming conditions. L-Histidine monohydrochloride monohydrate is obtained by fermentation of an isolated strain of Escherichia coli (ATCC-21318) derived by conventional mutation. The product is isolated from the fermentation broth, purified, including an ion-exchange procedure and therefore impurities from organic sources are not expected, de-colourized, concentrated, crystallized and dried. The European Food Safety Authority (EFSA) received a request from the Commission to evaluate the safety of the product for salmonids, the worker, the user, the consumer and the environment and to assess the product s bioavailability in order to add the product to the Annex of Council Directive 82/471/EC. With regard to bioavailability, studies on Atlantic salmon were performed using production parameter and muscle concentration of histidine as criteria. Based on muscle concentration data FEEDAP Panel concludes that the bioavailability of histidine from L- Histidine monohydrochloride monohydrate is as high as that of histidine from natural sources. There is limited scientific information on the use of excess dietary crystalline histidine in salmonids. However, a six month study on Atlantic salmon with dietary His levels up to 1.7 % (approximately two times higher than the dietary requirement of histidine for protein synthesis, but at about the recommended level for feeding practices), showed no adverse effects on fish growth or health. EFSA s FEEDAP Panel considers that since the product is an essential amino acid, potential safety concerns are expected to be primarily associated with the production method and the resulting purity of L-Histidine monohydrochloride monohydrate. It is not considered that the use of L-histidine in animal feed will increase human intake of that amino acid, and on the basis of the data available and considering the high degree of purity of L-Histidine monohydrochloride monohydrate obtained using a multistep physico-chemical purification process, the FEEDAP Panel does not expect a risk for the consumer. Specific studies concerning worker/user safety are not submitted, but due to the experience of the long term use of comparable products as food supplements and cosmetics ingredients, specific risks are not expected. However, workers and users should wear protective clothes as recommended by the applicant. The FEEDAP Panel concludes that the use of the product will not adversely influence the environment.

2 Opinion on the additive L-Histidine 2/10 Keywords: L-Histidine monohydrochloride monohydrate, His, EAA, fish feed, salmonids BACKGROUND Council Directive 82/471/EEC 1 establishes the rules governing the Community authorisation of certain products that act as direct or indirect protein sources and are manufactured by certain technical processes. This Directive defines the conditions that a substance/product should meet to get authorisation. Company Kyowa Kogyo Ltd. is seeking an authorisation of the product L-Histidine monohydrochloride monohydrate obtained by fermentation of an isolated strain of Escherichia coli and further isolation from the broth, de-colourisation, concentration, crystallisation and drying (98 % purity). This product belongs to the group 3.5 Amino acids and their salts (Table 1). Histidine is an essential amino acid that has been reported as powerful antioxidant. It is an important nutrient to control oxidative stress caused by temperature fluctuations. This amino acid was previously provided through the addition of blood meal to fish diets or the use of fishmeal from South America, which contain high levels of histidine. Currently, blood meal from ruminant origin is prohibited in fish feeding in the EU and only blood meal form other species is allowed. In addition, the South American supply for fish meal is limited. Consequently, the possibility to use this amino acid to supplement fish diets is an alternative that would allow maintaining adequate nutritional status in fish feeding. Table 1. Description of the product Product category: Trade name: Description: Target animal: Applicant: Type of request: 3.5 Aminoacids and their salts L-Histidine monohydrochloride monohydrate Amino acid obtained by fermentation of an isolated strain of Escherichia coli and further isolation from the broth, decolourisation, concentration, crystallisation and drying (98 % purity). Salmonids Kyowa Kogyo Ltd Authorisation not linked to a person for putting into circulation In accordance with the above legislation, the Commission received a request from Mr David Tennant on behalf of the company Kyowa Kogyo Ltd to authorise its product under the conditions set out in Table 2. Supporting data compiled by the company in a dossier accompanied that demand. This dossier is made available to the European Food Safety Authority directly by the company. 1 OJ L 213, , p.8

3 Opinion on the additive L-Histidine 3/10 Table 2. Annex entry, as proposed by the applicant The following is proposed to be added to point 3.5 of the Annex to Directive 82/471/EC Name of product group Name of product Designation of nutritive principle or identity of microorganism Culture substrate (identifications, if any) Composition characteristics of the product Animal species Special provisions 3.5 Histidin e L-Histidine monohydrochloride monohydrate technically pure C3H3N2-CH2-CH(NH2)- COOH HCl H2O L-Histidine monohydrochlorid e monohydrate 98% Salmonids Declarations to be made on the label and packaging of the product: -The name L-Histidine monohydrochloride monohydrate - L-Histidine monohydrochloride monohydrate and moisture contents -Animal species

4 Opinion on the additive L-Histidine 4/10 TERMS OF REFERENCE In view of the above, the Commission asks the European Food Safety Authority to issue an opinion on the safety for the target animals, the worker, the user, the consumer and the environment and the bioavailability of L-Histidine monohydrochloride monohydrate when used under the conditions set out in Table 2 above, taking into account the background and the information submitted by the applicant in the dossier. ASSESSMENT 1. Introduction The company has requested authorisation of its product L-Histidine monohydrochloride monohydrate (called L-Histidine HCl H2O in this document) an essential amino acid, to be incorporated in feed for salmonids. The product is obtained from a fermentation process which uses a strain of Escherichia coli derived from E. coli ATCC by conventional mutation. The fermentation medium used is a mixture of glucose syrup, ammonium sulphate, corn steep liquor and minerals. Histidine (His) is isolated from the fermentation broth, acidified, purified (including an ion exchange procedure) and de-colourised. After addition of hydrochloric acid the solution is concentrated and L-Histidine HCl H2O is crystallized and dried. The product is sieved through a 2 mm sifter, however, particle size distribution data were not provided. The chemical formula of the product is: C3H3N2-CH2- CH(NH2)-COOH. HCl. H20, molecular weight = , the CAS number is The product was shown to contain 74 % of L-histidine, equivalent to approximately 100 % L- Histidine HCl H2O 2. The purity of the product is claimed to be higher than 98 %, the analysis of three batches indicated values between %. The content of heavy metals (arsenic, lead cadmium and mercury) in the product using Atomic Absorption Spectrophotometry (AAS), Inductively Coupled Plasma Mass Spectrometry (ICP-MS, for As, Pb and Cd) and Cold Vapour Atomic Fluorescence Spectrophotometry (CV-AFS) are given in Table 4. Arsenic and mercury concentrations were below the detection limits for the methods used, trace amounts of cadmium were detected using ICP-MS, whereas there were disparities among the methods with regards to the lead concentration. Other impurities from organic sources are not to be expected due to the purification steps in the production process. Table 3. Heavy metal concentrations (impurities) in the product L-Histidine HCl H2O as mg kg -1 (data for 3 batches per method) As Pb Cd Hg AAS 1 <0.1 <0.05 <0.01 <0.01 ICP-MS/CV-AFS 2 < <0.002 < Supplementary dossier, Appendix F 2 Supplementary dossier, Appendix G ICP-MS = Inductively Coupled Plasma Mass Spectrometry, CV-AFS = cold vapor atomic fluorescence spectrometry The stability of the product in feed was tested at 8ºC for 18 months, and the losses were found to be low and comparable to the stability of His in endogenous His-rich feed (the histidine losses were 0.9 % and 6 % for L-Histidine HCl H2O supplemented diet and L-His rich diet from natural sources, respectively). However, usual conditions for testing stability 2 Supplementary dossier Appendix C

5 Opinion on the additive L-Histidine 5/10 would be shorter storage time at higher temperature. L-Histidine HCl H2O was not found to be hygroscopic following storage for 7 days at 25 C and 75 % humidity. The analytical method for the determination of L-His concentrations in feed involves the use of the Community methods for analysing amino acids (Commission Directive 98/64/EC 3 ). This method is used for routine feed quality control. 2. Bioavailability in fish Histidine is one of ten indispensable amino acids in fish nutrition, of which only the L-form of the amino acids are constituents of protein. Under pracitical farming conditions inclusion levels of histidine up to 1.7% of the diet for Atlantic salmon (Salmo salar L.) have been shown to be beneficial in preventing cataract formation (Bjerkås and Sveier, 2004; Breck et al. 2003) Two feeding trials with Atlantic salmon were submitted. A nine weeks growth trial in marine conditions was conducted using increasing supplementation levels of L-Histidine HCl H2O to a fish meal based diet. A control feed with 1.05 % His was supplemented with L- Histidine HCl H2O to contain 1.3 %, 1.5 % and 1.7 % His, respectively. A second control diet contained 1.7 % His from South American fish meal naturally rich in His. All diets supported satisfactory growth 4. Production parameters were not significantly improved by supplementation with the product, nor were they dose-dependent. Fish given the feed containing naturally high His had significantly higher growth than fish fed the control feed, the three groups given diets supplemented with L-Histidine HCl H2O had intermediate growth. The control feed and supplemented feeds contained soyameal and corn gluten, which reduce the metabolizable energy in the diet, whereas the feed based on South American fishmeal did not. The apparent digestibility of His increased with increasing L-Histidine HCl H2O supplementation ( %), and calculated partial His digestibility (i.e. added His) was between 95 and 100 %. The total His apparent digestibility was higher in L-Histidine HCl H2O supplemented diets than in the two control diets with naturally derived His at low and high levels. Muscle His reflected the dietary total His regimes (His + L-Histidine HCl H2O), with a span of 20 % increment. The bioavailability of L-Histidine HCl H2O in Atlantic salmon was also examined in a 13 weeks trial in marine conditions with fish fed the same supplemented diets as in the first trial. Muscle total His was recorded regularly, and showed a dose-response relationship after 7 weeks Conclusion on bioavailability Bioavailability studies with amino acids using production parameters as in the above experiments should be designed such that the amino acid in question is offered at different concentrations below the requirement. Also the test diets fed should have a comparable composition. Because both postulates were not fulfilled in the experiments on Atlantic salmon, FEEDAP Panel had to base conclusions on the parameter histidine in muscle tissue. It is concluded that the bioavailability of histidine from L-Histidine HCl H2O is at least as high as of His from natural sources. 3 O. J. L 257, 19/09/1998 P Main dossier, Appendix D

6 Opinion on the additive L-Histidine 6/10 3. Safety to the target species Toxicity studies with essential amino acids like histidine can not be designed along the lines of conventional toxicity experiments since the appearance of amino acid imbalances at higher dosages will dramatically restrict the desired margin of safety. There is limited scientific information on the effects of elevated dietary crystalline His in salmonids. However, a six month study on Atlantic salmon, which included both fresh water and marine conditions, with dietary His levels up to 1.7 % (approximately two times higher than the requirement of histidine for protein synthesis), showed no adverse effects on growth or health (Breck, 2004). It can be noted that elevated dietary histidine has been found to reduce levels of tissue zinc and copper due to increased mineral urine excretion and suppression of hepatic metallothionein-1 expression in rats (Aoyama et al., 1992, Aoyama and Cato, 2000). Trends of liver iron and zinc reductions were also observed in Atlantic salmon fed elevated dietary His up to 1.8 % (Breck et al., 2003). These findings may relate to the metal chelation properties described by Glover and Hogstrand (2002) of histidine. However, up to 1.7 % dietary His is recommended in normal feeding practices (Bjerkås and Sveier, 2004; Breck, 2004), 4. Safety - Studies on laboratory animals 4.1. Mutagenicity Mutagenicity testing was conducted according to the Guideline for genetic toxicity studies 5. Two batches of L-Histidine HCl H2O were tested in a reverse mutation test using Escherichia coli WP2 uvra which is a DNA-repair-proficient strain that is used routinely to test for DNA-cross-linking effects, one of the many ways in which mutations can occur. As that strain requires histidine to grow whereas the mutated reverted form does not, the test was performed after the strain was preincubated with L-Histidine HCl H2O. Dose levels of to 5 mg/plate were tested using or not rat S9 metabolic activation. No doubling of the number of revertant colonies compared with the negative control was observed in any trial. Whereas the two positive controls, (2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide and 2- aminoanthracene) increased the number of revertant colonies by 10 and 20 times, respectively. L-Histidine HCl H2O proved to be non-mutagenic in this specific test, however the test strain chosen was not the most appropriate Toxicological studies Sub-chronic oral toxicity study A rat diet supplemented with 10 % L-histidine expectedly retarded growth and resulted in moderate mortality within two months (Harper et al., 1970). A 13-week feeding study was performed using groups of 10 male and 10 female F344 rats fed diets containing 0, 0.31, 0.62, 1.25, 2.5 and 5 % L-Histidine HCl H2O produced by another company (Ikezaki et al., 1994). Feed consumption and growth were measured weekly along the experimental period. Haematological and serum biochemical tests as well as extended gross and histopathological examinations were performed at slaughter. Results were analysed statistically. No mortality occurred during the treatment period. When compared to control animals, lower growth rates and feed consumption were observed in the males of the 5 % group 5 Supplementary dossier as second response, Appendix A

7 Opinion on the additive L-Histidine 7/10 only. Haemoglobin volume and haematocrit increased significantly in the males of the 5 % group only. No serum biochemical abnormalities were observed in males of any treatment group whereas a significant increase of blood urea nitrogen was observed in the females of the 1.25 and 5 % groups and of creatinine in the females of the 5 % group. A significant increase in organ weight was observed for kidney in the males of the 2.5 and 5 % groups, for testis in the males of the 5 % group and for kidney in the female of the 5 % group. Gross pathological examination did not reveal anomalies whereas in histopathological analysis sperm granulomas in the epididymis were found in 5 of the 10 males of the 5 % group. In addition, mild histological changes were noted in the kidney, testis and spinal cord, but such changes did not appear in a dose dependent manner or in an extremely small number of animals. The authors attributed the negative effects on growth and feed consumption of the highest L-Histidine HCl H2O dose to the overdosage of His and subsequent amino acid imbalance. As His is a major component of haemoglobin, they hypothesized that the haematological changes might be related to an increase of haematocrit. The increase of kidney weight without gross or histopathological abnormality associated with the increase of blood urea nitrogen would suggest an effect of excessive amino nitrogen. Sperm granula formation in this study is suggestive of impairment of epididymis duct epithelium but without definite evidence. The FEEDAP Panel supports the authors conclusion that the maximum tolerable dose of L-Histidine HCl H2O was 2.5 % in the diet Chronic oral toxicity/carcinogenicity study A long term toxicity and carcinogenicity study (104 weeks) was performed using groups of 50 male and 50 female F344 rats fed diets containing 0, 1.25 and 2.5 % L- Histidine HCl H2O from another company, with a purity of 99.8 %. Feed consumption and growth were measured weekly during the first 13 weeks then every 4 weeks. After 104 weeks all surviving animals were given control diet for a further 3 weeks and then sacrificed. Blood was sampled for haematological determinations and gross examination was performed. Moribund and dead animals were autopsied. Histological examination of tumours was performed (Ikezaki et al., 1996). Significant depression of body weight was observed in the 2.5 % groups only for the week and week periods for the males and females respectively. Animals in the 2.5 % groups of both sexes showed slightly shorter mean survival times although these were not statistically significant. Increases in red blood cell count, haemoglobin value, haematocrit and platelet count were observed only in male rats of the 2.5 % group. The overall incidences of tumours in each group were 100 % in males and % in females. There were no significant differences between the control and treated groups of both sexes in overall tumour incidences. In all male groups, tumours of the testes were the most frequent, followed by lesions in the adrenal, haematopoietic organs and pituitary. All testicular tumours were benign interstitial cell tumours, the most frequently encountered spontaneous tumour in F344 rats. Sperm granulomas which were encountered in the subchronic toxicity study (4.2.1.) were not found in any group in the present study. The FEEDAP Panel concludes that this study gives weak evidence that L-Histidine HCl H2O is not carcinogenic in F344 rats of either sex. 5. Safety for the consumer As a general principle FEEDAP Panel considers conventional toxicology studies to be inappropriate for testing pure substances which are dietary nutrients (essential dietary nutrients). Such substances have a physiological concentration which is optimum for health and performance. Dietary intakes of such substances which lead to amounts which are significantly below or above that which is optimum for health and performance will inevitably cause a physiological imbalance and consequent adverse effects. This principle

8 Opinion on the additive L-Histidine 8/10 applies to substances where the purity is well established, with the source and method of production sufficiently well characterised for reassurance that no toxic contaminants will be present in the product. The testing appropriate to such substances will need to be judged on a case-by-case basis but in circumstances where the use of the substance leads to no increase in human intake there is no requirement for further toxicity data. Where there are major impurities, or the source is poorly characterised some evidence of safety from toxicological studies will be required. The testing programme would need to be designed to cover all likely areas of potential consumer risk. Where toxicological studies are already available due account will be taken of the data, but conduct of new studies for evaluation of the safety of such products should form part of a clear defined strategy relevant to assessing consumer safety. Justification for omitting studies of key effects from any submission should be made by the applicant as part of this defined strategy. In the particular case of L- Histidine HCl H2O this is confirmed by the lack of effects in the studies provided by the applicant: i) a study showing the lack of mutagenicity in one end point, ii) a sub-chronic toxicity study that indicated the additive was devoid of adverse effect in the rat for doses up to 2.5 % in the diet, iii) a chronic toxicity/carcinogenicity study that showed the lack of adverse effect of the additive in the rat at doses up to 1.25 % in the diet. In both sub-chronic and chronic studies adverse effects observed for the highest doses were attributed to a His imbalance. On the basis of the data available and considering the high degree of purity of L- Histidine HCl H2O obtained using a multistep physico-chemical purification process, the FEEDAP Panel does not expect a risk for the consumer. 6. Safety for the worker and user The proposed formulation for the product is as a purified, powdered form (particle size >2 mm), however, no data is provided regarding the particle size distribution. A draft manufacturer s safety data sheet is provided in the dossier and gives recommendations regarding occupational exposure control (the use of a mask, gloves, goggles, head cover and long-sleeved protectional clothing). The manufacturer claims that the product is not expected to cause skin or eye irritation. This is not supported with experimental data. Histidine is a normal constituent of plant and animal tissue. Technically pure L-histidine HCl is widely available as a food supplement and histidine is listed in the International Cosmetic Ingredient Dictionary and Handbook where it is included in cosmetics for hairand skin conditioning. Hence there is opportunity for oral and dermal exposure to Histidine HCl and the petitioner is unaware of any adverse effects, thus concludes that it is unlikely to represent a significant occupational health hazard. 7. Safety concerning the environment L-histidine is an essential amino acid and a natural component of animals and plants. Histidine from L-Histidine HCl H2O would replace histidine from natural sources and would therefore not lead to an increase of dietary histidine levels. Because synthetic histidine is absorbed at least as well as natural histidine sources, no increased excretion of histidine will occur. So, the use of L-Histidine HCl H2O as a feed additive is not expected to represent a risk to the environment. CONCLUSIONS AND RECOMMENDATION L-Histidine HCl H2O is a source of synthetic histidine able to replace histidine from natural sources. It is produced by fermentation of E. coli ATCC modified by conventional

9 Opinion on the additive L-Histidine 9/10 methods, and contains 74 % L-Histidine. Impurities from organic sources are not expected due to several specific purification steps during production process. Histidine from L-Histidine HCl H2O was shown to be at least as bioavailable in Atlantic salmon (Salmo salar) as His from natural sources. Under practical salmon feeding conditions, the minimum requirement may be exceeded by about 2.25 times (1.7 % in the diet) which probably reflects the practical upper limit. EFSA s FEEDAP Panel considers that since the product is an essential amino acid, potential safety concerns are expected to be primarily associated with the production method and the resulting purity of L-Histidine monohydrochloride monohydrate. It is not considered that the use of L-histidine in animal feed will increase human intake of that amino acid, and on the basis of the data available and considering the high degree of purity of L- Histidine HCl H2O obtained using a multistep physico-chemical purification process, the FEEDAP Panel does not expect a risk for the consumer. Specific studies concerning worker/user safety are not submitted, but due to the long term use of comparable products as food supplements and cosmetics ingredients, specific risks are not expected. However, workers and users should wear protective clothes as recommended by the applicant. The FEEDAP Panel concludes that the availability of the product will not adversely influence the environment because (i) the product replaces natural histidine sources, (ii) Histidine from the product is highly absorbed and (iii) faecal histidine is rapidly degraded in the environment. Recommendation to the Commission The annex entry for the product should contain in the fifth column (composition and characteristics of the product) the percentage of histidine content. DOCUMENTATION PROVIDED TO EFSA 1. Original L-Histidine monohydrochloride monohydrate dossier for use in animal nutrition by Kyowa Hakko Kogyo dated 28 April Response to the first request for complementary information 3. Response to the second request for complementary information dated 3 December Supplementary dossier as a response to Member State questions (NL) REFERENCES Aoyama, Y., Mori, M., Hitomi-Ohmura, E. and Yoshida, A Effects of dietary excess histidine and varying levels of copper on the metabolism of lipids and minerals in rats. Biosci. Biotechnol. Biochem. 56, Aoyama, Y. and Cato, C Suppressive effect of excess dietary histidine on the expression of hepatic metallothionein-1 in rats. Biosci. Biotec. and Biochem., 64, Bjerkås, E. and Sveier, H The influence of nutritional and environmental factors on osmoregulation and cataracts in Atlantic salmon (Salmo salar L.). Aquaculture 235,

10 Opinion on the additive L-Histidine 10/10 Breck, O., Bjerkås, E., Campbell, P., Arnesen, P., Haldorsen, P. and Waagbø, R Cataract preventative role of mammalian blood meal, histidine, iron and zinc in diets for Atlantic salmon (Salmo salar L.). Aquaculture Nutrition, 9, Breck, O Histidine Nutrition and Cataract development in Atlantic salmon, Salmo salar L. Dr. Scient. Thesis, University of Bergen - NIFES, Bergen, Norway. Glover, C.N. and Hogstrand, C Amino acid modulation of in vivo intestinal zinc absorption in freshwater rainbow trout. J. Exp. Biol., 205, Harper, A.E., Benevenga, N.J. and Wohlhueter, R.M Effects of ingestion of disproportionate amounts of amino acids. Physiol. Rev. 50, Ikezaki, S., Nishikawa, A., Furukawa, F., Imazawa, T., Enami, T., Mutsui, M. and Takahashi, M., week subchronic toxicity study of L-Histidine monohydrochloride in F344 Rats. Eisei Shikensho Hokoku, 112, Ikezaki, S., Nishikawa, A., Furukawa, F., Enami, T., Mutsui, M., Tanakmaru, Z., Kim, H-C., Lee, I-S. Imazawa, T. and Takahashi, M Long-term toxicity/carcinogenicity study of L-Histidine Monohydrochloride in F344 rats. Food and Chemical Toxicology 34, SCIENTIFIC PANEL MEMBERS Arturo Anadón, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Gerhard Flachowsky, Anders Franklin, Jürgen Gropp, Anne-Katrine Lundebye Haldorsen, Ingrid Halle, Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Pieter Wester and Wilhelm Windisch. ACKNOWLEDGEMENT The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to thank Professor Rune Waagbo for his contribution to the draft opinion.