The EFSA Journal (2004) 121, 1-13

Transcription

1 The EFSA Journal (2004) 121, 1-13 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the Commission related to the preliminary assessment of the safety of Nitarsone (4-nitrophenylarsonic acid), as a feed additive in accordance with Regulation (EC) N 178/2002 and Regulation (EC) N 1831/2003, article 15. SUMMARY (Question N EFSA-Q ) Adopted on 28 October 2004 Nitarsone (4-nitrophenylarsonic acid 4-NPA) is used to control blackhead disease, a debilitating protozoal infection in turkeys. The additive is due for evaluation to comply for a provisional authorisation for a maximum period of five years. The European Commission asked the European Food Safety Authority to make a preliminary evaluation of the safety of 4-nitrophenylarsonic acid, and its metabolites when it is used as feed additive for animal nutrition. The additive is not yet in use in the European Union. Nitarsone (a chemically synthesized organoarsenical) at a dose level of mg of 4-NPA kg -1 of feed is recommended to be administered to turkeys from 2 weeks until 12 weeks of age. The very limited data derived from efficacy studies indicate a possible margin of safety to be four times the recommended dose (750/187.5 mg 4-NPA kg -1 ). However, the FEEDAP Panel expressed their concern to set a preliminary margin of safety value because the studies are more than 40 years old, the number of animals per group is low, the treated animals are all infected, the duration of the experiments are short and the effects of 4-NPA to gut flora are not explored. No data are available concerning the metabolism of 4-NPA in turkeys. The metabolic pathways of 4- NPA in the laying hen indicate that the arsanilic acid is the major metabolite excreted with no release of inorganic arsenic. However, the methods used to investigate 4-NPA metabolism are questionable in terms of specificity and sensitivity. No data are given concerning the nature of tissue residues. No data have been supplied on the metabolism of 4-NPA in laboratory animals. Residue studies of total arsenic in turkey tissues indicate that arsenic concentrations increase following exposure of the animals to 4-NPA but decline to control levels after a 9-day withdrawal period. However, no data are given concerning the kinetics of 4-NPA residues in turkey tissues. The limited data available give no indication of genotoxicity or carcinogenicity but no data at all are available on developmental and reproductive effects. A NOEL can not be firmly established but it would be approximately 2 mg 4-NPA kg -1 bw day -1, based on the chronic toxicity study in rats. However, there is evidence that dogs are maybe more sensitive to 4-NPA and a properly conducted study in dogs may give a lower NOEL. Additional studies would be needed in order to further refine the assessment of the consumer safety. Since a NOEL cannot be confidently established from the available toxicity data an acceptable daily intake (ADI) value cannot be determined. The data available does not allow the FEEDAP Panel to propose maximum residue limit (MRL) values. No validated control methods are supplied for 4-NPA in premixes and feedstuffs. Validated control methods would be required for any marker residue when this can be established. Key words: 4-nitrophenylarsonic acid, 4-nitarsone, arsenical compounds, blackhead disease, turkey, animal safety, consumer safety, ADI, MRL.

2 Opinion on 4-nitrophenylarsonic acid 2/13 Table of contents 1. Introduction Structural formula Mode of action Stability Control methods Studies on target species Tolerance tests on target species/animal categories Other effects on target species Effects on gut flora Conclusion Metabolism and residue studies Laboratory animals Studies on laboratory animals Acute Oral Toxicity Genotoxicity In vivo rat liver UDS assay Subchronic (90-day) oral toxicity studies day study in rats day study in dogs Carcinogenicity and chronic oral toxicity studies Two- year feeding study in CD-1 mice ( ) Two-year feeding study in Swiss Webster mice (1964) Two year feeding study in Long-Evans Rats ( ) Two year feeding study in Sprague-Dawley Rats (undated) Two year feeding study in dogs (1964) Neurotoxicity studies (indication for related compounds) Reproductive Toxicity Studies, including developmental toxicity Multigeneration Study Developmental Studies Conclusion on carcinogenicity and chronic toxicity Safety evaluation for the human consumer Proposal of the acceptable daily intake (ADI) Proposal of the maximum residue limits (MRLs)...11 Conclusions and Recommendations...12

3 Opinion on 4-nitrophenylarsonic acid 3/13 BACKGROUND Council and Parliament Regulation (EC) N 1831/ foresees in Article 15 the possibility that the Commission may provisionally authorise, for a maximum period of five years, an additive to ensure the protection of animal welfare; in view of the above the European Commission requested an opinion, in accordance with Regulation (EC) N 178/2002 and Regulation (EC) N 1831/2003, article 15. Nitarsone is an organic arsenic molecule, currently used in Canada and the USA, to prevent histomoniasis (blackhead disease), however the situation in the European Union is quite different where the additive is not authorised. Taking into account the urgency of this matter for turkey farming a preliminary data package on nitarsone has been presented to the Commission, for use as feed additive to prevent the histomoniasis following the conditions proposed in the dossier. TERMS OF REFERENCE The Commission asks the European Food Safety Authority to make a preliminary evaluation of the safety of 4-nitrophenylarsonic acid, and its metabolites when it is used as feed additive for animal nutrition following the conditions in the dossier. ASSESSMENT This preliminary assessment will focus on the safety of animals and consumers only. A complete assessment would be required when seeking for a ten-year period authorisation. 1. Introduction Nitarsone (4-nitrophenylarsonic acid 4-NPA) is used to control blackhead disease (histomoniasis) in turkeys. The feed additive is manufactured by blending 4-NPA with calcium carbonate, rice hulls and mineral oil. Data confirm that moisture is below 1% w/w and inorganic arsenic (as trivalent arsenic) below 1%. No indication on particle size <10 µm is given. The final concentration of 4-NPA in the additive is mg 4-NPA kg -1 and it is recommended to be administered in feed from 2 until 12 weeks of age. Nitarsone (CAS number ) is a non-chiral chemically synthesized organoarsenical where arsenic is in its pentavalent form. 4-NPA synthesis is described in detail but spectral characterization of the product is not given. Specifications of the chemicals used for the manufacture of this compound are given. A minimal purity of 95% is used in the preliminary data package but no information is given concerning 4-NPA impurities (maximum 5%) with the exception of inorganic arsenic which limit is fixed to <1%. According to the dose level of 4-NPA proposed for turkey feeds, the additional contribution of residual inorganic arsenic to the current arsenic load in feeds would be negligible. 1 E.C.O.J. n L 268 of , p. 29

4 Opinion on 4-nitrophenylarsonic acid 4/ Structural formula 4-NPA has a molecular weight of and contains 30.33% of arsenic. The physical properties of 4-NPA indicate a melting point of C (decomposition). 4-NPA is a strong acid with pka values of 3.1 (molecular ion) and 7.8 (mono-anion). 2 It is weakly soluble in cold water and moderately soluble in warm water. However, solubility figures were not provided. O OH As OH NO 2 4-nitrophenylarsonic acid (4-Nitarsone) 1.2 Mode of action No data on the mode of action has been supplied in the preliminary data package. 1.3 Stability The stability of the additive during storage (shelf life) has been monitored at 25 C and 37 C at ambient humidity for 12 months and at 25 C for 24 months (only two batches). Results available after 12 months storage at both temperatures indicate a gradual and slow downward trend which falled inside the specification determined in the preliminary data package (50 ± 5 % of 4-NPA). After 24 months of storage one batch was outside of the specifications (44%). 3 Information on the degradation products was not provided. The stability of 4-NPA in premixes during manufacture and storage was not supplied. Very limited data indicate that 4-NPA is stable in feeds during manufacture and storage. 1.4 Control methods A HPLC method has been validated for the determination of 4-NPA in the additive but no method has been described in the preliminary data package for the determination of 4-NPA either in premixes and feeds or as a residue in tissues, keeping in mind the hypothesis that 4-NPA would be retained as the marker residue. A spectrophotometric method for the determination of arsenic in chicken tissues has been applied on the range of expected concentrations (0.025 to 0.5 mg kg -1 in the muscle, 0.1 to 5 in the liver, 0.2 to 2 in the kidney and 0.05 to 1 in the skin/fat). The method was validated on the concentration range studied. The determination involves an ashing step in which inorganic arsenic but also its organic forms including 4-NPA and related metabolites are mineralized to arsenic hydride which is reacted with a colouring reagent. Depending on the tissue, the limits of detection 2 Volume 1 of 5, Reference Volume 1 of 2. Section 2.3

5 Opinion on 4-nitrophenylarsonic acid 5/13 (LODs) and quantification (LOQs) varied between 0.03 to 0.1 mg kg -1 and 0.03 respectively. to 0.2 mg kg -1 Capillary electrophoresis has been developed and applied for 4-NPA (Sun, et al., 2002 and 2003). 2. Studies on target species 2.1 Tolerance tests on target species/animal categories Studies dedicated to evaluate tolerance were not provided. Some data on efficacy were in the dossiers which were used to extract a very preliminary margin of safety. Mc Guire and Morehouse (1952) treated 4 to 6 weeks old infected poults with 4-NPA in water and in feed. The group sizes are between 6-13 poults per treatment. All animals died if treated with 500 mg L -1 of 4-NPA in water or 2000 mg kg 1 in feed. Five out of eight animals died if treated with 1000 mg kg -1 of 4-NPA in feed. The average number of animals per group was 13 and 6 respectively. Salsbury, 4 studied the effect of 4-NPA administered in water and in feed to 3 to 6 weeks old infected turkeys. The average number of animals per group was 5. Animals received 540 mg L -1 died of toxicity of 4-NPA. Doses from 324 to 405 mg L -1 did not cause the death of the animals, however the growth was low. All poults which received 1500 or 2000 mg of 4-NPA kg -1 of feed died of toxicity. Animals treated with 1000 mg of 4-NPA kg -1 in feed showed a lower growth rate but treatment with 750 mg kg -1 resulted in normal growth. 2.2 Other effects on target species One day old turkey poults which were not infected by histomonad were exposed (15 weeks females, 24 weeks males) to copper (120 mg kg -1 Cu as Cu2O) and 4-NPA (187.5 mg kg -1 feed). The data indicates that the incidence of aortic rupture was greater in the presence of 4-NPA and was reduced by one-half by copper supplementation (Guenthner, et al, 1978). In the European Union, the copper supplementation, at these levels, is not allowed by current legislation. 2.3 Effects on gut flora No data were available in the preliminary data package. 2.4 Conclusion The very limited data derived from efficacy studies indicate a possible margin of safety to be four times the recommended dose (750/187.5 mg 4-NPA kg -1 ). However, the FEEDAP Panel expressed their concern to set a preliminary margin of safety value because the studies are more than 40 years old, the number of animals per group is low, the treated animals are all infected, the duration of the experiments are short and the effects of 4-NPA to gut flora are not explored. 4 Volume 1 of 5. Reference 5.

6 Opinion on 4-nitrophenylarsonic acid 6/ Metabolism and residue studies A study of the metabolic fate of 4-NPA in laying hens using a single oral dose of [ 14 C-1]-4- nitrophenylarsonic acid has been provided in the preliminary data package. 5 Different trials were conducted with three to five times higher dosage (50 mg per bird) than the exposure that would result from feed supplementation using the recommended dose. The work was carried out using the best methodology available during the 50 s. The main conclusions are the following: i) the metabolic balance indicates that on the third day post-administration, 60 to 90% of the dose is excreted and a complete excretion is observed after nine days; the presence of significant amounts of radioactivity in the tissues and bile indicates that 4-NPA is absorbed to a certain extent, ii) unchanged 4-NPA and its reduction product 4-aminophenylarsonic acid (arsanilic acid) were the main compounds found in the excreta, resulting from the reduction of the nitro group. iii) the production of arsenoxides as well as the cleavage of the molecule and production of inorganic arsenic was very limited or absent. One study (1964) 6 described the elimination of 4-NPA in humans (5 males) when given per os a single 20 mg dose. Two dogs received the same dosage in feed. In humans, the results indicated that 29 to 63% of the total organic arsenic ingested was excreted in the urine. A similar figure was observed in dogs (45 and 57%). Using thin layer chromatography it was demonstrated that unchanged 4-NPA and arsanilic acid were the major metabolites in the urine of both species. In a study 7 (author and date not specified, protocol insufficiently detailed) the increase of total arsenic contents in chicken edible tissues was investigated. Chickens were fed diets supplemented with 125 mg of 4-NPA kg -1 for 30 consecutive days and followed by a withdrawal period of 1, 3, 5 and 7 days. The volumetric method used for the determination of total arsenic (including 4-NPA and metabolites) in tissues (Cassil and Wichman, 1939) gave elevated standard deviations (up to 100% for background levels of 0.1 to 0.3 mg kg -1 ). In these conditions, after a metabolic equilibrium was reached (2 to 5 days) no cumulative process was observed during the 30-day administration period and the arsenic concentration in tissues declined to the background level after a 5-day withdrawal period. In a study reported in 2004, 8 groups of five male turkeys were selected for tissue collection (liver and breast muscle) among control animals fed a standard diet and animals receiving for 7 consecutive weeks a feed supplemented with the additive (187.5 mg 4-NPA kg -1 feed) followed by a 9-day withdrawal period. Total arsenic measured in turkey tissues indicate that arsenic concentrations increase following exposure of the animals and after a 9-day withdrawal period, arsenic concentrations in all tissues declined below 0.2 mg kg -1 (<0.6 mg 4-NPA kg -1 wet tissue) with the exception of one muscle sample (0.22 mg kg -1 ). A review of data from the literature concerning the metabolism of 4-hydroxy-3-nitrophenylarsonic acid (roxarsone) has been supplied but it is not considered further as these results cannot be extrapolated to 4-NPA. No data have been supplied concerning the kinetics of 4-NPA residues in turkey tissues. 2.6 Laboratory animals. No data have been supplied on the metabolism of 4-NPA in laboratory animals. 5 Volume 1 of 5, Reference Volume 1 of 5, Reference Volume 1 of 5. Reference Supplementary data. August 2004.

7 Opinion on 4-nitrophenylarsonic acid 7/ Conclusion In the laying hen, a single dose of 4-NPA is absorbed at an unknown extent and metabolized relatively slowly but completely excreted after 9 days. It is metabolized mainly to arsanilic acid resulting from the reduction of the nitro-group, the production of arsenoxides as well as the cleavage of the molecule and production of inorganic arsenic being very limited or absent. However, the methods used are questionable in terms of specificity and sensitivity. Although no data have been supplied on the metabolism of 4-NPA in turkeys, it can be anticipated that the metabolic fate of 4-NPA in another galliforme would not be essentially different from that in the laying hen but this remains to be demonstrated. No data have been supplied on the metabolism of 4-NPA in laboratory animals. Residue studies of total arsenic in turkey tissues indicate that arsenic concentrations increase following exposure of the animals to the additive but decline to control levels after a 9-day withdrawal period (< 0.6 mg 4-NPA kg -1 wet tissue). However, no data are given concerning the kinetics of 4-NPA residues in turkey tissues. 3. Studies on laboratory animals 3.1 Acute Oral Toxicity The only acute toxicity data included in the dossier are in the form of a summary table 9 with no details of experimental procedure or dates of studies. The LD50 after oral dosages in rats is 130 mg of 4-NPA kg 1 bw (range between mg of 4- NPA kg 1 bw) The LD50 for intraperitoneal exposure is 46.5 mg of 4-NPA kg 1 bw (range between mg of 4-NPA kg 1 bw). 3.2 Genotoxicity There is only one genotoxicity study included in the dossier In vivo rat liver UDS assay A study was completed in 2004, 10 compliant with both GLP and with current international guidelines. The study showed a good response to the positive control material and no indication of any effect of NPA at the maximum tolerated dose of 125 mg 4-NPA kg -1 bw day -1. At this dose there was no sign of significant cytotoxicity in the hepatocytes of 4-NPA treated animals thus the results are considered a valid assessment of genotoxicity. Although the study provided is an in vivo mammalian test, and thus gives considerable reassurance on the lack of genotoxicity of 4-NPA, further data would be required for full assessment. These data would consist of either a second in vivo study in a different tissue or at least two in vitro studies measuring different end-points. 9 Volume 1 of 5, Reference Supplementary data. Dated 2004.

8 Opinion on 4-nitrophenylarsonic acid 8/ Subchronic (90-day) oral toxicity studies day study in rats This study 11 was conducted in 1959 and consisted of six groups of six rats of each sex fed diets ranging from 0 to 200 mg 4-NPA kg -1 respectively. A further group was fed control diet in amounts restricted to match the dietary intake of the 200 mg 4-NPA kg -1. Body weight was measured at weekly intervals and blood samples were taken for haematology weekly for the first month and monthly thereafter. At the study termination all animals were brought to necropsy and a range of organ weights and histology samples taken. Apart from the low numbers of animals per group the study is adequately conducted. The only effects seen from this study were in the kidneys of males given the highest two doses. These effects consisted of severe degenerative lesions (classified as more severe than seen in controls and lower dose groups) plus fibrosis in the highest dose animals The NOEL was therefore concluded to be equivalent to 125 mg 4-NPA kg -1. Following the calculations applied in the preliminary data package 12 this equates to an intake of approximately 12.5 mg 4-NPA kg -1 bw day day study in dogs This study was conducted in 1959 and consisted of five groups of two dogs of each sex that were given diets ranging from 0 to 250 mg 4-NPA kg -1 dry feed respectively, for 1 week. Following repeated signs of toxicity (vomiting) at these doses for 6 days, the concentrations were reduced to 25 to 100 mg 4-NPA kg -1 dry feed, respectively. Body weights were recorded weekly throughout the study and blood samples were taken for haematology at weekly intervals for the first month and monthly thereafter. Rectal temperatures were taken twice weekly. All animals were brought to necropsy at study termination. Only adrenals and thyroids were weighed but a full range of tissues were taken for histopathology. Body weight was decreased by treatment in the highest dose group and gain was reduced compared with controls in the group given 50 mg 4-NPA kg -1 dry feed. The weight gain of the other two groups was not notably different from controls. Three deaths occurred during the study but none were related to treatment. The highest dose group showing no effects was the one which received 37.5 mg 4-NPA kg -1 dry feed, however due to the changes of dose during the study, the state of health and variability of the animals a NOEL cannot be calculated. Considering the shortcomings of the study, caution is suggested when using these results in any ADI calculation. This study is poorly conducted and cannot be used as a reliable indicator of the toxicity of 4-NPA. However, it does provide an indication that the dog may be more sensitive to 4-NPA than the rat. 3.4 Carcinogenicity and chronic oral toxicity studies Two- year feeding study in CD-1 mice This study 13 tested 4-NPA at dietary concentrations ranging from 0 to 400 mg kg -1 using groups of 80 individually housed mice of each sex at each dose level and 160 of each sex for the controls. (A further group was treated with a positive control material but this is not considered relevant to this assessment). The details provided on this study are little more than an overview, with no detailed tabulated study data. At the highest dose it is said that there was a reduction in body weight gain and an effect on overall mortality. A full necropsy with preservation of tissues was carried out 11 Volume 1 of 5, Reference Volume 1 of 5, page Volume 1 of 5. Reference 18

9 Opinion on 4-nitrophenylarsonic acid 9/13 wherever possible and abnormal tissues were examined microscopically. The report on histopathology is presented as a separate reference 14 and does not flag up any treatment-related effects on incidence of either benign or malignant tumours. Due to deficient reporting, this study is of very limited value but provides some reassurance that there is no carcinogenic effect of the test substance 4-NPA Two-year feeding study in Swiss Webster mice Groups of 50 mice of each sex, housed in cages of five to ten animals per cage were fed diets ranging from 0 to 50 mg 4-NPA kg -1 for two years. 15 Animals were monitored daily and food intake and body weight was recorded weekly. There was no adverse effect on growth or mortality of the animals during the study. A slightly higher incidence of mammary tumours in the highest dose group was explored in detail and specific analysis including comparison with contemporary controls from other studies suggests that this difference is not an effect of treatment. The NOEL from this study is at the highest dose of 50 mg kg -1 4-NPA which can be considered roughly equivalent to 7.5 mg 4-NPA kg -1 bw day -1, however there were no measurements of indices relevant to toxicity and the main value of this study is further reassurance of the lack of carcinogenic effect of 4-NPA Two year feeding study in Long-Evans Rats Groups of 60 rats of each sex were individually housed and given diets ranging from 25 to 250 mg 4-NPA kg -1 ; a group of 120 rats of each sex received control diet. 16 Body weight and food intake were recorded weekly. Blood was taken for haematology at 2, 8, 14, 20 and 24 months of treatment from 6 of sex from each group. Urinalysis was carried out at 3, 9, 14, 20 and 24 months of treatment. A full necropsy was carried out on all animals where possible and tissues collected for histological examination. Organs were weighed at the necropsy of animals brought to necropsy at the end of the study. At the highest dose there was decreased body weight gain and reduced survival compared with controls. Haematology and urinalysis showed no changes that were related to treatment. Some differences were seen in organ weights but these are difficult to interpret from animals at this stage of life and should be considered mainly in relation to the histopathology. The histopathology examination is reported in a separate reference 17 and no difference is seen in the incidence rates of tumours in treated and control groups. Enlarged bile ducts are reported to be present more frequently in the livers of rats from the 125 and 250 mg 4-NPA kg -1 groups but no tabulation of incidence data is given. The NOEL from this study is thus at the dietary concentration of 50 mg 4-NPA kg -1 which from the study data would appear to correspond with an intake during the last 18 months of treatment of between 2 and 2.5 mg 4-NPA kg -1 bw day -1. This study is a reasonably full investigation of chronic toxicity and probably provides the closest indication of a NOEL as well as further reassurance of the lack of carcinogenicity of 4-NPA. 14 Volume 1 of 5. Reference Volume 2 of 5, Reference Volume 2 of 5, Reference Volume 2 of 5, Reference 22

10 Opinion on 4-nitrophenylarsonic acid 10/ Two year feeding study in Sprague-Dawley Rats Very limited data are provided for this study. 18 Groups of 50 rats of each sex individually housed were fed diets containing 0, 25 or 50 mg 4-NPA kg -1 for 2 years; dose levels were originally higher, but they have been reduced due to toxicity. There is no indication of when this reduction took place. Body weight and food intake were recorded weekly but no other measurements were made on the animals during their life. All animals were brought to necropsy where possible and tissues preserved for microscopic examination. Organ weights were recorded on animals dying during the last 6 months of the study. The body weight gain of high dose rats was lower than that of controls and mortality in this group was higher than controls. No differences were seen in food intake or organ weight data. Tumour incidences were similar in all groups. Bile duct hyperplasia was seen in many of the high dose animals of both sexes but not in any of the animals from the lower dose group. The lowest dose used in this study 25 mg 4-NPA kg -1 is the NOEL but the effects at the higher dose may in part be due to the much higher initial dose given to these animals. It would be unsafe to use these data to derive a NOEL expressed in mg 4-NPA kg -1 bw day -1. Since the dose given to the rats in this study was reduced due to toxicity at an unspecified time this study is not considered suitable for further consideration Two year feeding study in dogs The report 19 provided is a very brief summary of the study making independent assessment impossible. Groups of three dogs of each sex were fed diets ranging from 0 to 25 mg 4-NPA kg -1 for 133 weeks (Initially doses were set at higher levels and were reduced in two stages due to evident toxicity; the doses stated were given from week 15 onwards). Body weight was recorded weekly and 3-monthly examinations were carried out covering haematology, urinalysis and kidney and liver function tests. A necropsy was carried out on all animals with samples preserved for histopathological examination. The reported histopathology data seem incomplete in that they show only one high dose male to have been examined and this is recorded to have died early in the study. Since no data are presented on mortality further analysis or comment on this study seems to offer little value hence no conclusion could be reached. Since the dose given to these animals was reduced twice during the course of the study, due to unexpected toxicity, it is not considered to be of value in the review of the safety of 4-NPA Neurotoxicity studies (indication for related compounds) The FEEDAP Panel express their concern on the lack of neurotoxicity studies in the dossier; due to the nature of the compound. 3.6 Reproductive Toxicity Studies, including developmental toxicity Multigeneration Study A 3-generation study on Roxarsone 20 was included in the preliminary data package, however no further consideration was given, as it does not relate to the active ingredient, 4-NPA. 18 Volume 2 of 5. Reference Volume 2 of Reference Volume 2 of 5. Reference 26

11 Opinion on 4-nitrophenylarsonic acid 11/ Developmental Studies Rats A fertility and developmental study on Roxarsone 21 was provided; however no further consideration was given, as it does not relate to the active ingredient, 4-NPA.. Rabbits A fertility and developmental study on Roxarsone 22, 23 was provided; however no further consideration was given, as it does not relate to the active ingredient, 4-NPA. 3.7 Conclusion on carcinogenicity and chronic toxicity An in vivo mammalian genotoxicity test gives considerable reassurance on the lack of genotoxicity of 4-NPA. Further data are required for full assessment consisting of either a second in vivo study in a different tissue or at least two in vitro studies measuring different end-points. Long term studies did not show any indication of carcinogenic potential of 4-NPA. Despite deficiencies in study design and the limitations of evaluation resulting from presentation of incomplete study reports, the FEEDAP Panel can conclude that there is no evidence for carcinogenic activity of 4-NPA. Hepatic toxicity in the rat in the form of enlarged bile ducts is the key chronic toxic effect in the long-evans rat study giving a NOEL of approximately 2 mg 4-NPA kg -1 bw day -1 but without the full report of the histological evaluation of this study for independent assessment, the NOEL can only be used as an indicator of the order of magnitude of the likely NOEL. The highest dose used in the rat 90-day study was equivalent to half the highest dose of the chronic study and thus must be considered inadequate by current standards. It should also be noted that although a 90-day study in dogs is inadequate it provides an indication that dogs may be more sensitive to 4-NPA, and thus a lower NOEL may be established from a properly conducted study in dogs. 4. Safety evaluation for the human consumer 4.1 Proposal of the acceptable daily intake (ADI) Due to absence of a complete set of toxicological data, it prevents the establishment of an acceptable daily intake ( ADI). Moreover even if an ADI could be established, the absence of data on the metabolic pathways of 4-nitrophenylarsonic acid in the laboratory animals and the turkey would preclude any assessment of the adequacy of the toxicological evaluation of residues in turkey tissues. 4.2 Proposal of the maximum residue limits (MRLs) As no ADI can be set and no human exposure to 4-NPA has been established, no proposal for an MRL can be made. 21 Volume 3 of 5. Reference Volume 5 of 5. Reference Volume 5 of 5. Reference 29

12 Opinion on 4-nitrophenylarsonic acid 12/13 CONCLUSIONS AND RECOMMENDATIONS From the assessment of the the preliminary data package available for 4-NPA the FEEDAP Panel draws the following conclusions and recommendations: SAFETY FOR TARGET SPECIES The very limited data derived from efficacy studies indicate a possible margin of safety to be four times the recommended dose (750/187.5 mg 4-NPA kg -1 ). However, the FEEDAP Panel expressed their concern to set a preliminary margin of safety value because the studies are more than 40 years old, the number of animals per group is low, the treated animals are all infected, the duration of the experiments are short and the effects of 4-NPA to gut flora are not explored. SAFETY FOR THE CONSUMER No data are available concerning the metabolism of 4-NPA in turkeys. The metabolic pathways of 4- NPA in the laying hen indicate that the arsanilic acid is the major metabolite excreted with no release of inorganic arsenic. However, the methods used to investigate 4-NPA metabolism are questionable in terms of specificity and sensitivity. No data are given concerning the nature of tissue residues. No data have been supplied on the metabolism of 4-NPA in laboratory animals. Residue studies of total arsenic in turkey tissues indicate that arsenic concentrations increase following exposure of the animals to the additive but decline to control levels after a 9-day withdrawal period. However, no data are given concerning the kinetics of 4-NPA residues in turkey tissues. An in vivo mammalian genotoxicity test gives considerable reassurance on the lack of genotoxicity of 4-NPA. Further data are required for full assessment consisting of either a second in vivo study in a different tissue or at least two in vitro studies measuring different end-points. Long term studies did not show any indication of carcinogenic potential of 4-NPA. Despite deficiencies in study design and the limitations of evaluation resulting from presentation of incomplete study reports, the FEEDAP Panel can conclude that there is no evidence for carcinogenic activity of 4-NPA. Hepatic toxicity in the rat in the form of enlarged bile ducts is the key chronic toxic effect in the Long-Evans rat study giving a NOEL of approximately 2 mg 4-NPA kg -1 bw day -1 but without the full report of the histological evaluation of this study for independent assessment, the NOEL can only be used as an indicator of the order of magnitude of the likely NOEL. The highest dose used in the rat 90-day study was equivalent to half the highest dose of the chronic study and thus must be considered inadequate by current standards. It should also be noted that although a 90-day study in dogs is inadequate it provides an indication that dogs may be more sensitive to 4-NPA, and thus a lower NOEL may be established from a properly conducted study in dogs. In summary, a NOEL cannot be confidently established from the available toxicity data and an acceptable daily intake (ADI) value cannot be determined. Even if an ADI could be established, the absence of data on the metabolic pathways of 4-NPA in the laboratory animals and the turkey would preclude any assessment of the adequacy of the toxicological evaluation of residues in turkeys tissues; consequently an MRL can not be established.

13 Opinion on 4-nitrophenylarsonic acid 13/13 MONITORING Validated control methods are supplied for 4-NPA in the feed additve but no methods have been described in the preliminary data package, to identify 4-NPA in premixes and feedstuffs. Validated control methods would be required for any marker residue when this can be established. RECOMMENDATIONS In order to enable a more comprehensive assessment of 4-NPA, it is recommended that the following data is provided: Additional tolerance test, Microbiological safety of the additive to the target species, Data on the metabolic pathways of 4-NPA in turkeys and laboratory animals, Data on tissue residues in turkeys A completed toxicity data set. DOCUMENTATION PROVIDED TO EFSA 1. Nitarsone Dossier. December Supplementary data REFERENCES Cassil, C.C. and Wichman, H.J A rapid volumetric method for determining arsenic. Journal of the Association of Official Agricultural Chemists 22, Guenthner, E., Carlson, C. and Emerick, R Copper salts for growth stimulation and alleviation of aortic rupture losses in turkeys. Poultry Science 57, 5, Mc Guire, W.C. and Morehouse, N.F Chemotherapy studies of histomoniasis. Poultry Science. Volume XXXI, 4, Sun, B., Macka, M. and Haddad, P Separation of organic and inorganic arsenic species by capillary electrophoresis using direct spectrophotometric detection Electrophoresis 23, Sun, B., Macka, M. and Haddad, P Trace determination of arsenic species by capillary electrophoresis with direct UV detection using sensitivity enhancement by counter or co-electroosmotic flow stacking and a high sensitivity cell. Electrophoresis 24, SCIENTIFIC PANEL MEMBERS Arturo Anadón, Margarita Arboix Arzo, Georges Bories, Paul Brantom, Joaquim Brufau de Barbera, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Gerhard Flachowsky, Anders Franklin, Jürgen Gropp, Anne-Katrine Haldorsen, Ingrid Halle, Alberto Mantovani, Kimmo Peltonen, Guido Rychen, Pascal Sanders, Amadeu Soares, Pieter Wester and Wilhelm Windisch.