Scientific Opinion on the safety of Hostazym X as a feed additive for poultry and pigs 1

Transcription

1 EFSA Journal 2015;13(1):3969 SCIENTIFIC OPINION Scientific Opinion on the safety of Hostazym X as a feed additive for poultry and pigs 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Hostazym X is an enzyme preparation of xylanase produced by a non-genetically modified strain of Trichoderma citrinoviride. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) issued an opinion on the safety and efficacy of the product as a feed additive for poultry, piglets (weaned) and pigs for fattening. The Panel could not conclude on the safety for the target species, the consumer and the user because the presence of genotoxic activity in the fermentation product could not be excluded. The applicant provided new experimental data to support the safety of the fermentation product used to formulate the additive. The FEEDAP Panel concludes that the fermentation product used to formulate Hostazym X is unlikely to present a genotoxic hazard. Taking also into consideration the findings of the previous opinion, Hostazym X is considered safe for target animals and consumers. With regards to user safety, the previous conclusion that the additive should be considered a potential skin and eye irritant, and a potential skin and respiratory sensitiser, applies. European Food Safety Authority, 2015 KEY WORDS zootechnical additives, digestibility enhancers, endo-1,4-beta-xylanase, poultry, pigs, safety 1 On request from the European Commission, Question No EFSA-Q , adopted on 10 December Panel members: Gabriele Aquilina, Vasileios Bampidis, Maria De Lourdes Bastos, Lucio Guido Costa, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Fernando Ramos, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Enzymes, including Paul Brantom, Noël Dierick, Ingrid Halle and Pasquale Mosesso, for the preparatory work on this scientific opinion. Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on the safety of Hostazym X as a feed additive for poultry and pigs. EFSA Journal 2015;13(1):3969, 10 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

2 SUMMARY Following a request from the European Commission, EFSA s Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of Hostazym X (endo-1,4-beta-xylanase) as a feed additive for poultry and pigs. This additive is a preparation of endo-1,4-beta-xylanase produced by a non-genetically modified strain of Trichoderma citrinoviride. The liquid and solid formulations of this product are authorised as feed additives for chickens for fattening, turkeys for fattening, laying hens and piglets (weaned). In 2010, the applicant requested that its use in feed be extended to poultry, piglets and pigs for fattening as a zootechnical additive, in the functional group of digestibility enhancers. In 2013, the FEEDAP Panel adopted an opinion on the safety and efficacy of Hostazym X. The set of toxicological studies evaluated during the assessment consisted of a bacterial reverse mutation assay, a chromosome aberration test, an in vivo micronucleus test, an in vivo comet assay and a sub-chronic oral toxicity study, which were performed with the enzyme concentrate used in the manufacture of the additive. The results of the in vitro chromosomal aberration test and of the in vivo comet assay indicated the presence of genotoxic activity. Therefore, the FEEDAP Panel could not conclude on the safety of the additive for the target species, the consumer and the user because the presence of genotoxic activity in the fermentation product could not be excluded. To support the absence of toxigenic activity and the safety of the fermentation product used to formulate the additive, the applicant submitted new in vitro and in vivo experimental studies. In the three in vitro chromosome aberration studies performed in human lymphocytes, neither genotoxicity nor cytotoxicity was reported. Two in vivo comet assays, conducted in different laboratories, were negative for the induction of DNA breakage and reported no local cytotoxicity. Similar negative results were reported in a third in vivo comet assay that, according to the applicant s declaration, was conducted on the same batch that showed positive results in the first in vivo comet study. Therefore, the positive result reported in the previous in vivo comet study is to be considered an isolated result, that is not reproducible even when the same product batch was tested. The FEEDAP Panel concludes that the fermentation product used to formulate Hostazym X is unlikely to present a genotoxic hazard. Taking also into consideration the findings of the previous opinion, Hostazym X is considered safe for target animals and consumers. However, with regards to user safety, the previous conclusion that the additive should be considered a potential skin and eye irritant, and a potential skin and respiratory sensitiser, applies. EFSA Journal 2015;13(1):3969 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Background as provided by the European Commission... 4 Terms of reference as provided by the European Commission... 4 Assessment Introduction Safety for the target species, consumer and user In vitro chromosome aberration studies First study Second study Third study In vivo comet assay studies First study Second study Third study... 8 Discussion... 9 Conclusions... 9 Documentation provided to EFSA... 9 References EFSA Journal 2015;13(1):3969 3

4 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Regulation (EC) No 1831/2003 establishes rules governing the Community authorisation of additives for animal nutrition and in particular, Article 9 defines the terms of the authorisation by the Commission. The applicant HUVEPHARMA is seeking a Community authorisation of its of endo-1,4-beta xylanase (Hostazyme X), to be used as zootechnical additive (Table 1). Table 1: Description of the substance Category of additive Zootechnical additives Functional group of additive Digestibility enhancers Trade name - Description endo-1,4-beta xylanase produced by Trichoderma citrinoviride (IM SD135) Target animal category Poultry, piglets and pigs for fattening Applicant HUVEPHARMA Type of request Update opinion On 31 th January 2013, the Panel on Additives and Products or Substances used in Animal Feed of the European Food Safety Authority ( Authority ) gave an opinion on the safety of the product. It was concluded that owing to the genotoxic activity present in the product, even if the results obtained in subchronic studies did not indicate any concerns for the consumers, the Panel was not able to conclude on the safety for the consumers. Since the additive was already authorised and because of contradictory results in different genotoxicity studies, in some cases performed following old scientific approach, the Commission gave the possibility to the applicant to submit complementary information to complete the assessment on safety to allow a revision of that opinion. The Commission has now received an additional dossier from the applicant 4 on endo-1,4-beta xylanase (Hostazyme X) with supplementary information, concerning the genotoxic studies of the additive. The data generated by the applicant and compiled in the above-mentioned supplementary reports have been sent directly to the Authority. 5 TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In view of the above, the Commission requests the European Food Safety Authority to deliver an opinion on the safety of endo-1,4-beta xylanase (Hostazym X) as zootechnical additive submitted by HUVEPHARMA. 4 Huvepharma NV, Uitbreidingstraat 80, 2600 Antwerp, Belgium. 5 EFSA Dossier reference: FAD EFSA Journal 2015;13(1):3969 4

5 ASSESSMENT 1. Introduction The additive Hostazym X is a preparation of endo-1,4-beta-xylanase (xylanase; EC ) produced by a non-genetically modified strain of Trichoderma citrinoviride Bisset (IMI SD135), formerly identified as Trichoderma longibrachiatum. Liquid and solid formulations of this product are authorised as feed additives for chickens for fattening, turkeys for fattening, laying hens and piglets (weaned). In 2010, the applicant requested that the additive be re-evaluated when used in these species/categories and that its use in feed be extended to poultry for fattening or laying and pigs for fattening as a zootechnical additive, in the functional group of digestibility enhancers. The FEEDAP Panel adopted, in 2013, an opinion on the safety and efficacy of the product as a feed additive for poultry and pigs (EFSA FEEDAP Panel, 2013). The FEEDAP Panel could not conclude on the safety for the target species, the consumer and the user because the presence of genotoxic activity in the fermentation product could not be excluded. The applicant has submitted new experimental data to support the safety of the fermentation product used to formulate the additive. 2. Safety for the target species, consumer and user In 2013, the FEEDAP Panel adopted an opinion on the safety and efficacy of Hostazym X as a feed additive for poultry and pigs (EFSA FEEDAP Panel, 2013). The opinion included the characterisation of the product and of the production strain and an assessment of the safety of the additive for the consumers, users and the environment and the safety and efficacy for the target species. The set of toxicological studies evaluated during that assessment consisted of a bacterial reverse mutation assay, a chromosome aberration test, an in vivo micronucleus test, an in vivo comet assay and a sub-chronic oral toxicity study, which were performed with the enzyme concentrate used in the manufacture of the additive. In the 2013 opinion, the results of the in vitro chromosomal aberration test and of the in vivo comet assay indicated the presence of genotoxic activity. On the basis of these experimental studies, the FEEDAP Panel concluded that while a contribution of cytotoxicity to the observed induction of DNA breakage is possible, the presence in the fermentation product of genotoxic impurities cannot be excluded (EFSA FEEDAP Panel, 2013) and, therefore, it was not possible to conclude on the safety for the consumer and for the target species. Moreover, the FEEDAP Panel considered exposure of the user to the additive to be potentially hazardous. To support the absence of genotoxic activity in the fermentation product, the following in vitro and in vivo experimental studies have been submitted by the applicant In vitro chromosome aberration studies Three in vitro studies in human lymphocytes were submitted: two newly performed in 2013 and one performed in 1995, which was not submitted and assessed in the previous opinion First study The test item, Hostazym X concentrate, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments, in accordance with Organisation for Economic Co-operation and Development (OECD) Guideline 473 (revision 1997) 6. The study design was as follows: experiment 1: 4 hours exposure, followed by 18 hours recovery, with and without S9 mix; 6 Technical Dossier/Ref 3 Study EFSA Journal 2015;13(1):3969 5

6 experiment 2A: 4 hours exposure, followed by 18 hours recovery, with S9 mix, or 22 hours exposure (no recovery time), without S9 mix; experiment 2B: 22 hours exposure (no recovery time) without S9 mix. Cells were treated with the following concentrations of the test item: 0, , or μg/ml. In each experimental group, two parallel cultures were analysed. For each culture, 100 metaphases were evaluated for structural chromosomal aberrations, with the exception of the μg/ml Hostazym X concentrate cultures in experiment 2 in the absence and presence of S9 mix, in which 200 metaphases were evaluated, and the positive control in experiment 2A, in the absence of S9 mix, in which only 50 metaphases were evaluated. No cytotoxicity was observed up to the highest applied concentration in the absence and presence of S9 mix. In experiment 1, in the absence of S9 mix, two slight but statistically significant increases (2 % aberrant cells, excluding gaps) were observed after treatment with the intermediate concentrations ( and μg/ml), while no increase was observed at the highest concentration (5 000 μg/ml). It should also be noted that the increased values were within the range of the laboratory historical solvent control data ( % aberrant cells, excluding gaps). Therefore, these increases were considered to have no biological relevance. In experiment 2A, after 22 hours continuous treatment in the absence of S9 mix, a single statistically significant increase in chromosomal aberrations (4.3 % aberrant cells, excluding gaps) was observed after treatment with μg/ml, this value being slightly higher than laboratory historical control data ( % aberrant cells, excluding gaps). For this reason the experiment was repeated (experiment 2B). As this finding was not confirmed in experiment 2B, the finding noted in experiment 2A is considered to be spurious. In conclusion, the test item was considered not clastogenic. No increase in polyploid metaphases was observed in any experimental condition. Appropriate mutagens used as positive controls induced statistically significant clastogenicity Second study A second identical study, performed in the same laboratory and over the same period (March April 2013), was submitted by the applicant 7. The test item, Hostazym X concentrate, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments, in accordance with OECD Guideline 473 (revision 1997). The study design was as follows: experiment 1: 4 hours exposure, followed by 18 hours recovery, with and without S9 mix; experiment 2: 4 hours exposure, followed by 18 hours recovery, with S9 mix, or 22 hours exposure (no recovery time), without S9 mix. Cells were treated with the following concentrations of the test item: 0, , or 5000 μg/ml. In each experimental group, two parallel cultures were analysed. For each culture, at least 100 metaphases were evaluated for structural chromosomal aberrations. In experiment 1, in the absence of S9 mix, 400 metaphases were scored in the negative control group and all Hostazym X concentration groups owing to inhomogeneous data. No cytotoxicity was observed up to the highest applied concentration in any experimental condition. No clastogenicity was observed in the absence or presence of S9 mix. However, in experiment 1, in the absence of S9 mix, a single slight, but statistically significant, increase was observed after 4 hours treatment with μg/ml (2.8 % aberrant cells, excluding gaps). This value was within the range of the laboratory historical solvent control data ( % aberrant cells, excluding gaps). No similar increase was observed in experiment 2 after 22 hours treatment with μg/; thus, this finding was regarded as biologically irrelevant. In experiment 2, in the presence of S9 mix, a single increase slightly exceeding the range of 7 Technical Dossier/Ref 4 Study EFSA Journal 2015;13(1):3969 6

7 the laboratory historical solvent control data ( % aberrant cells, excluding gaps) was observed after treatment with μg/ml (3.5 % aberrant cells, excluding gaps). Since the increase was not statistically significant, and no increase was noted at the higher concentration levels, the finding has to be regarded as biologically irrelevant. No evidence of an increase in polyploidy was reported. Positive controls performed as expected Third study A third study on human lymphocytes in vitro reported negative results. This study was performed in 1995 and the experimental procedure was compatible with OECD Guideline 473 (1983 version). 8 According to a declaration provided by the applicant, the test item used in this study corresponds to the Hostazym X concentrate used in the other genotoxicity studies submitted. Cultured human lymphocytes were treated in duplicate with a range of three concentrations, up to a maximum of 5 mg/ml. The cells were treated 48 hours after culture initiation for 20 hours without S9 mix or for 3 hours with S9 mix, arrested in metaphase and harvested 20 hours after the start of the treatment. Two independent experiments were conducted. No change in the mitotic index was observed in any experimental condition. In the first experiment with S9 mix, a statistically significant increase in aberrant metaphases was reported only at the highest concentration, but this finding was not confirmed in the second experiment. A further experiment with a harvest time of 44 hours gave negative results. Positive controls gave the expected response in all the experiments In vivo comet assay studies The applicant proposed that the positive outcome reported in the previous in vivo comet assay study was an artefact due to residual test item in the analysed cells. Therefore, two further in vivo comet studies were submitted, in which, according to the applicant, more thorough washing of the organs was incorporated before cell isolation to avoid that residues of Hostazym X concentrate remained during further preparation of the cells. In addition, a third in vivo comet assay was conducted on the same batch that showed positive results in the first in vivo comet study. These three studies were performed before the publication of OECD Guideline 489 on the in vivo mammalian alkaline comet assay (26 September 2014); however, the experimental procedure followed was compatible with this guideline First study Hostazym X concentrate was assessed in the in vivo alkaline single cell gel electrophoresis analysis for its potential to induce primary DNA damage in cells prepared from the small intestine and stomach of Wistar WU rats 9. The test item was suspended in sterile water. Six male rats per group were treated twice orally with a dose of Hostazym X concentrate of 500, or mg/kg body weight (bw) 24 and 4 hours prior to preparation of the cells. The volume administered orally was 10 ml/kg bw. After the end of the treatment period, the animals were sacrificed and cells were isolated by mincing the small intestine tissue and scraping the stomach tissue. Clinical observation revealed ruffled fur at all tested dose levels. The frequency of this finding increased with dose level. The number of apoptotic and necrotic cells in the small intestine and stomach, determined per total nuclei for each animal, was negligible. In each experimental group, including the control group, 150 cells from the small intestine and 150 cells from the stomach of each of the six male rats were assessed for DNA damage. Compared with the corresponding vehicle-treated control rats, none of the rats treated with Hostazym X at any of the tested dose levels exhibited a biologically relevant or statistically significant increase in DNA damage according to the evaluated parameter (per cent tail intensity). In addition, the per cent tail intensities of the Hostazym X-treated animals were all within or slightly below the historical control data range 8 Technical Dossier/Ref 5 Study Technical dossier/ref 6 Study EFSA Journal 2015;13(1):3969 7

8 for the small intestine and stomach. Treatment with the positive control substance (25 mg/kg bw methyl methane sulfonate) led to a distinct and statistically significant increase in DNA damage Second study Hostazym X was tested for its potential to induce DNA damage in the stomach and duodenum of treated rats 10. Six male Sprague Dawley rats were treated twice, at intervals of 21 hours, by oral gavage with 125, 500 or mg/kg bw/day in purified water (treatment volume 10 ml/kg bw). Stomach and duodenum were sampled 3 hours after the second administration. Following treatment with Hostazym X at all dose levels, group mean per cent tail intensity and tail moment values for both tissues were similar to those of the concurrent vehicle control group, and all individual animals were considered comparable to the vehicle controls. There was no dose-related increase in the percentage of clouds or percentage of cells with halos in the stomach or duodenum following treatment, with vehicle control data for all tissues being comparable to the laboratory s historical data. No signs of toxicity were observed in any animals following administration of Hostazym X. In the positive control group (200 mg/kg bw ethyl methane sulfonate, dosed at 21 hours), mean comet parameters in both tissues were markedly increased compared with the concurrent vehicle control group. It was concluded that, under the conditions of this in vivo comet assay, Hostazym X did not induce DNA damage in the stomach or duodenum of rats treated at doses of up to mg/kg/day, the maximum dose currently recommended for the comet assay Third study The Panel concluded that the two new in vivo comet studies provided by the applicant were scientifically reliable, but found no obvious reason to reject the positive in vivo comet study assessed in the 2013 opinion. Therefore, the Panel considered the hypothesis that the difference in the outcome of the studies could reflect a change in the manufacturing process of the enzyme. The applicant stated that no changes to the fermentation and purification process had been introduced over the period during which the studies had been conducted. At the request of the Panel, the applicant performed a third in vivo comet assay, which was conducted (according to the applicant s declaration) on the same batch that had showed positive results in the first in vivo comet study. This is described below. The test item, Hostazym X concentrate, was assessed in the in vivo comet assay for its potential to induce primary DNA damage in cells prepared from the small intestine and stomach of rats 11. The test item was dissolved in sterile water. Six male rats per group were treated twice orally with a dose of 500, or mg/kg bw 24 and 4 hours prior to preparation of the cells. The volume administered orally was 10 ml/kg bw. At the end of the treatment period, the animals were sacrificed, and cells were isolated by mincing the small intestine tissue and scraping the stomach tissue. In the high-dose group, diarrhoea was observed in three males at 2 hours after the second application of the test item and in six males at 4 hours after the second application. No other sign of toxicity was observed. The number of nuclei from apoptotic or necrotic cells per total nuclei was determined for each animal as an indicator of the quality of the slide preparation. In the case of cells from the small intestine and stomach, the number of dead cells was negligible. In each experimental group, including the control group, 150 cells from the small intestine and stomach of all animals, i.e. six males per group, were assessed for DNA damage. At none of the tested dose levels was there a biologically relevant or statistically significant increase in DNA damage in the 10 Supplementary information January 2014/Appendix Supplementary information September 2014/Ref 2. EFSA Journal 2015;13(1):3969 8

9 cells of the treated animals, as determined by per cent tail intensity, compared with the vehicle-treated control rats. In addition, all per cent tail intensities of the Hostazym X concentrate-treated animals were within the historical control data range for the small intestine and stomach. For both tissues, values in vehicle controls were in the range of the historical control data, confirming the valid performance of the study. In the positive control group (25 mg/kg bw methyl methane sulfonate), there was a distinct and statistically significant increase in DNA damage, as detected by per cent tail intensity analysis. In conclusion, in three new in vivo comet assays, two oral doses of 500, or mg/kg bw Hostazym X concentrate did not induce any DNA damage in cells isolated from the small intestine or stomach of male rats. Therefore, Hostazym X concentrate is considered to be non-genotoxic in these three new in vivo alkaline single cell gel electrophoresis assays. DISCUSSION In its previous opinion (EFSA FEEDAP Panel, 2013), Hostazym X was negative in a bacterial reverse mutation assay. Clastogenicity was observed in an in vitro chromosome aberration test in Chinese hamster ovary cells, but only in the absence of metabolic activation, and was accompanied by slight to evident cytotoxicity. Negative results were reported in an in vivo micronucleus test in bone marrow, in which, however, no evidence of target exposure was reported. Three in vitro chromosome aberration studies performed in human lymphocytes were submitted by the applicant for the present opinion; no genotoxicity or cytotoxicity was reported. The in vivo comet assay using oral administration assessed in the 2013 opinion showed the induction of DNA breakage at the site of contact (glandular stomach and duodenum) and, at higher dosages, local cytotoxicity, as revealed by histopathological analysis. In three new in vivo comet assays, conducted in two different laboratories, Hostazym X did not induce DNA breakage and no local cytotoxicity was reported. It should be noted that one of these studies was performed with the same product batch which had shown positive results in the first in vivo comet study. The positive result reported in one of four in vivo comet studies is considered to be an isolated result, not reproducible even when the same batch was tested and, therefore, on the basis of the data set provided by the applicant, the fermentation product used to formulate Hostazym X is unlikely to present a genotoxic hazard for target animals, consumers and users. CONCLUSIONS The FEEDAP Panel concludes that the fermentation product used to formulate Hostazym X is unlikely to present a genotoxic hazard. Taking also into consideration the findings of the previous opinion, Hostazym X is considered safe for target animals and consumers. However, with regards to user safety the previous conclusion that the additive should be considered a potential skin and eye irritant, and a potential skin and respiratory sensitiser applies. DOCUMENTATION PROVIDED TO EFSA 1. Supplementary Information Dossier. October Submitted by HUVEPHARMA NV. 2. Supplementary Information Dossier. Supplementary information. January Submitted by HUVEPHARMA NV. 3. Supplementary Information Dossier. Supplementary information. September Submitted by HUVEPHARMA NV. EFSA Journal 2015;13(1):3969 9

10 REFERENCES EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Scientific Opinion on the safety and efficacy of Hostazym X (endo-1,4-beta-xylanase) as a feed additive for poultry, piglets and pigs for fattening. EFSA Journal 2013;11(2):3105, 19 pp. doi: /j.efsa EFSA Journal 2015;13(1):