(Question N EFSA-Q ) adopted on 06 July 2005

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1 Opinion of the Scientific Panel on Plant health, Plant protection products and their Residues on a request from EFSA related to the evaluation of pirimicarb SUMMARY OF OPINION (Question N EFSA-Q ) adopted on 06 July 2005 The PPR Panel 1 was requested to give an opinion on the assessment of acute risk to birds from the use of pirimicarb, a carbamate insecticide, which is used in wheat, and also on the use of a time quotient approach in such assessments. The PPR Panel concludes that the time quotient approach suggested by OECD (1996) is equivalent to the current EU first tier acute avian risk assessment using a TER 2, except that Annex VI to Directive 91/414/EEC 3 stipulates the use of a safety factor of 10 whereas OECD (1996) recognises the potential need for a safety factor but does not recommend a specific value for it. A detailed analysis would be required to assess whether the current EU safety factor of 10 takes appropriate account of all relevant issues, including those raised by the notifier in the case of pirimicarb (acuteness of exposure and the rate of absorption, distribution, metabolism and excretion) and the substantial variation in toxicity between species. The PPR Panel concludes that such an assessment would require substantial further work that is beyond the scope of this Opinion, and recommends that consideration be given to pursuing it as a separate initiative. In the meantime, the PPR Panel suggests that issues of the type raised by the notifier should be assessed case by case, as part of a refined, higher tier assessment. The PPR Panel assessed the risk to birds feeding on insects in cereal fields after treatment with pirimicarb. Based on the results of this assessment, the PPR Panel is of the opinion that even at the upper limit of credible exposures, birds feeding on insects in the field are unlikely to achieve a lethal dose of pirimicarb. Studies indicate that pirimicarb is rapidly absorbed, metabolised and eliminated by the animal. Pirimicarb exerts its toxic effects by inhibiting the enzyme acetylcholinesterase. This inhibition and the consequent signs of toxicity are reversible within hours from onset. Moreover, these effects require a high blood level of pirimicarb that occurs after direct dosing of the animal (gavage) but not when pirimicarb is incorporated into the diet to provide a similar dose. Therefore, the TER of 4.77 based on the LD50 4 determined from gavage of birds was considered to overestimate the risk. On the other hand, the shortterm TER of 92.5 based on the LD50 estimated from a 5-day study with pirimicarb incorporated in the diet also does not closely represent the pattern of exposure expected in the field. Therefore, the PPR Panel carried out a refined assessment taking account of expected feeding rates of wild birds, the toxicokinetic and toxicodynamic characteristics of pirimicarb, and the reduction of exposure due to food avoidance. The framework used in this opinion may be applied to other compounds and situations, on a case-by-case basis. Key words : insecticide, pirimicarb, carbamate, time quotient approach, TER, safety factors, bird, toxicity, acute risk, reversibility, dietary study, acetylcholinesterase, metabolism, food avoidance. 1 PPR Panel: Panel on Plant health, plant protection products and their residues 2 TER: Toxicity-exposure ratio 3 OJ No L 230, , p.1 4 LD50: Lethal dose; the dose at which 50 % of the test organisms die 1 of 21

2 TABLE OF CONTENTS Summary of Opinion... 1 Table of contents... 2 Background... 3 Terms of reference Assessment question 1: the time quotient approach according to OECD (1996) Equivalence of time quotient approach and toxicity-exposure ratio (TER) Safety factors for use in acute risk assessment for birds Conclusions and Recommendations on Question Assessment question 2: assessment of the acute risk to birds from the use of pirimicarb Introduction Toxicity of carbamates: inhibition of acetylcholinesterase (AChE) Characteristics of inhibition of AChE by carbamates Toxicological data Data on birds: gavage and dietary exposures Bobwhite quail Mallard ducks Conclusions on data on birds Supporting information Data on mammals Conclusions on data on mammals Feed intake, avoidance response, rate of absorption, distribution, metabolism and excretion, and reversibility of effects in acute risk assessment for birds General approach Assessment for pirimicarb Choice of species and estimation of feeding rate Estimation (assumption) of LD Estimation of Avoidance Threshold Dose (AVT) Estimation of avoidance delay time (AVD) Estimation of concentration of pirimicarb on small insects Estimation of half-life for elimination of pirimicarb Estimation of dose and consideration of uncertainties Summary of refined assessment General comments on available data to assess the acute risk for birds Conclusions and Recommendations on Question Documentation provided to EFSA...18 References...19 Scientific Panel members...20 Appendix I Derivation of Equation of 21

3 BACKGROUND 5 The insecticide pirimicarb is the ISO 6 common name for 2-dimethylamino-5,6- dimethylpyrimidin-4-yl dimethylcarbamate (IUPAC) 7. It is a specific aphicide that acts by inhibiting the acetylcholinesterase enzyme. The use considered is the product Pirimor (a WG formulation containing 500 g/kg pirimicarb) for use on wheat with maximum 2 applications of g pirimicarb per ha. The first application is foreseen between BBCH and the second application is foreseen between BBCH The risk to birds is assessed in the DAR 9 on pirimicarb by comparing the LD50 value to the daily food consumption (wet weight) using residue data outlined in EPPO 10 (1994) and not following the new guidance document on risk assessment for birds and mammals (SANCO 11, 2002a). An acute toxicity study is available with bobwhite quail (LD50 = 20.9 mg/kg bw 12 ) and mallard (LD50 = 28.5 mg/kg bw). Emesis occurred in the acute toxicity study on mallard. However looking at the similar overall acute and dietary toxicity profile of bobwhite quail and mallard with pirimicarb, it was considered appropriate by the RMS 13 to discount the acute mallard study and use the lower LD50 of 20.9 mg/kg bw from the study with bobwhite quail. The representative use is wheat at BBCH This is before the grain has ripened and the ears of wheat are unlikely to be attractive to birds. It is also considered to be beyond the stage where there would be significant grazing of wheat or weeds by birds. Therefore the risk was assessed for insectivorous birds. The resulting acute TER-value is 4.77 so the Annex VI trigger value of 10 from the Directive 91/414/EEC is breached indicating a high acute risk to birds. Consequently a refinement of the acute risk assessment was performed. The notifier has argued that the acute oral LD50 is not the most appropriate endpoint to be used in this risk assessment since exposure of small birds will be over a period of time (i.e. dietary). They consider it unlikely that a bird will be able to ingest sufficient treated food to consume a lethal dose within a single day. This is in line with the time quotient >1 scenario put forward in the Report of the SETAC 14 /OECD Workshop on Avian Toxicity Testing (OECD 1996). The RMS (UK) considered this argument valid but stated that it does not fully consider the issue of interspecies sensitivity. The RMS considers that a TER of 4.77 gives some margin of safety but more information needs to be considered. Therefore they compare the dietary endpoint for bobwhite quail which they converted into daily dose according to SANCO (2002a) and compare it with the acute ETE 15 calculated according to EPPO (1992) which gives a TER of 90. In a similar way they compared the 20 week NOEC to this acute ETE which gives a TER of 6. Based on these refinements the RMS considers the acute risk to birds to be addressed. This time quotient approach was discussed during the EPCO Expert meeting and it was decided to forward this question to the PPR Panel. 5 Background delivered by EFSA s PRAPeR sector (coordination of the pesticide risk assessment peer review of active substances) 6 ISO: International Organization for Standardization 7 IUPAC: International Union of Pure and Applied Chemistry 8 BBCH: Scale for standard codification of growth stages of mono- and dicotyledone plants 9 DAR: Draft Assessment Report 10 EPPO: European and Mediterranean Plant Protection Organisation 11 SANCO: Directorate General for Health and Consumer Affairs 12 bw: body weight 13 RMS: Rapporteur Member State 14 SETAC: Society of Environmental Toxicology and Chemistry 15 ETE: Estimated Theoretical Exposure 16 EPCO: EFSA Peer Review Co-ordination 3 of 21

4 TERMS OF REFERENCE The Scientific Panel on Plant Health, Plant Protection Products and their Residues (PPR Panel) of EFSA is asked for an opinion on: 1) the time quotient approach according to OECD (1996) 2) the assessment of the acute risk to birds from the use of pirimicarb, taking into account the available information including: metabolism studies, reversibility of effects, type of effect observed in the acute and dietary studies, time to onset of effects observed in the acute/dietary studies. 1 Assessment question 1: the time quotient approach according to OECD (1996) 1.1 EQUIVALENCE OF TIME QUOTIENT APPROACH AND TOXICITY-EXPOSURE RATIO (TER) This question refers to the time quotient approach proposed by a SETAC/OECD workshop on avian toxicity testing (OECD, 1996). The workshop report defines time quotient as the time required (days) to consume an LD50 when feeding on contaminated food (p. 23 in OECD, 1996), i.e. the LD50 divided by the daily exposure. Therefore the time quotient is exactly equivalent to the toxicity-exposure ratio (TER) that is currently used in acute avian assessments under Directive 91/414/EEC. The SETAC/OECD workshop proposed that the time quotient can be used to identify the appropriate toxicological test for avian assessments. Specifically, it proposed that the avian assessment should be based on the acute oral test when the time quotient is 1 day or less (i.e. TER 1), and that in such cases consideration might be given to conducting additional acute oral toxicity studies (pp. 23, 103 and 112, OECD 1996). This is equivalent to using TER 1 as a trigger for requiring refinement of the acute avian risk assessment. The workshop also proposed that, when the time quotient exceeds 1 day (i.e. TER > 1), the avian risk assessment should be based instead on subacute (i.e. 5-day) dietary toxicity tests. This would imply that no further consideration of acute risk is required when the time quotient based on a single acute oral LD50 exceeds 1. Therefore, the basic time quotient approach proposed by OECD (1996) is equivalent to using TER 1 as the criterion for requiring further consideration of acute avian risk, instead of the criterion of TER < 10 that is specified in Annex VI of Directive 91/414/EEC. 1.2 SAFETY FACTORS FOR USE IN ACUTE RISK ASSESSMENT FOR BIRDS The SETAC/OECD workshop recognised that concerns exist about extrapolating from three standard test species (mallard, bobwhite quail and Japanese quail) to the wide range of species that may be exposed in the field (p. 120, OECD 1996). The workshop proposed several approaches to this problem including testing of further species, the use of field studies and postregistration monitoring, or the use of larger safety factors. 4 of 21

5 As recognised by the RMS, the argument of the notifier in the case of pirimicarb is equivalent to using the basic time quotient approach without any safety factor to allow for extrapolation of toxicity between species. In fact, the time quotient may be much lower (implying higher risk) for some of the species exposed in the field, because LD50s can vary by one or two orders of magnitude between species for the same pesticide (e.g. Luttik and Aldenberg, 1997). A statistical analysis of this variation (such as that of Luttik and Aldenberg) could therefore contribute to selecting an appropriate safety factor for use with the time quotient. However, such a safety factor should also take account of other factors and uncertainties affecting the level of protection provided by the assessment. As shown by the preceding paragraphs, the time quotient is exactly equivalent to a TER, so adopting a safety factor other than 10 for the time quotient would imply deviating from Annex VI of Directive 91/414/EEC. This would be a major change in regulatory practice, which would require substantial justification. The notifier gives two arguments that should be considered if a change of safety factor was to be contemplated, either in the specific case of pirimicarb or more generally. First, they argue that the acute oral LD50 is not the most appropriate endpoint since the dietary exposure of small birds to residues on small insects occurs gradually over a period of time, rather than almost instantaneously as occurs with an acute oral dose. This is a general argument that could potentially be proposed for many pesticides, except those where birds could ingest a lethal dose in a single bolus of food (e.g. some granular or bait formulations, treated seeds, and sprayed formulations of pesticides with high acute toxicity). The notifier s second argument is that the rapid absorption, distribution, metabolism and excretion (ADME) of pirimicarb, and the rapid reversibility of its effects, decreases the likelihood that the effective internal dose experienced by a bird will reach a lethal level. This argument could also be proposed for other rapidly-cleared compounds. The PPR Panel agrees that the issues underlying both of the notifier s arguments the acuteness of the exposure, and the rate of ADME 17 and of reversibility of effects do influence risk to birds. The implied question here is whether these issues justify changing the safety factor and the way the toxicity studies are used in acute avian risk assessments. Answering this question is made more difficult by the fact that the original basis of the current safety factor is not explained in Directive 91/414/EEC or its Annexes, nor in the relevant EU Guidance Documents (SANCO 2002a,b), nor as far as the PPR Panel can ascertain anywhere else. Therefore, it is not possible to check whether the issues raised by the notifier were considered when setting the current safety factor. A new assessment would be required to determine whether those issues are appropriately accounted for by the current safety factor of 10, or whether a change is justified. Such an assessment would need to consider not only the issues raised by the notifier, but also all other factors that have significant influence on the protectiveness of the acute avian assessment, including the wide variation in toxicity between species as mentioned above 18, and the overall level of protection desired. Two contrasting approaches for conducting such an assessment were suggested previously in an Opinion by the Scientific Committee on Plants (section in SCP, 2002). Briefly, they were (1) modelling and uncertainty analysis to provide a theoretical estimate of the level of protection provided by the first tier assessment, and (2) calibrating the level of protection by comparing the first tier assessment with empirical data on field impacts, e.g. the database of Mineau (2002). An analysis of the latter type has been conducted and summarised in the proceedings of a workshop (Mineau and Whiteside, in press) but not peer-reviewed or published in detail. An analysis of this type might provide a reasonable basis for reassessing the current approach to 17 ADME: absorption, distribution, metabolism and excretion 18 Examples of additional issues that would need to be taken into account include use of the LD50 as the measure of toxicity (rather than more protective endpoints such as the LD10 or the NOEC which would take account of adverse effects below the level of 50% mortality), and the conservatism of the exposure assessment. 5 of 21

6 acute avian risk assessment and the issues raised by the notifier, but this would require detailed evaluation (including an assessment of the relevance of the field impacts data to EU conditions) and is beyond the scope of the current opinion. The PPR Panel concludes that further work would be required to assess the scientific basis for a general change to the safety factor used for acute avian risk. In the meantime, the PPR Panel suggests that the influence on risk of the acuteness of the exposure, the rate of ADME and of reversibility of effects should be assessed case by case as part of a refined, higher tier assessment. This is considered in question 2 for the case of pirimicarb. 1.3 CONCLUSIONS AND RECOMMENDATIONS ON QUESTION 1 The PPR Panel concludes that the time quotient approach suggested by OECD (1996) is equivalent to the current EU first tier acute avian risk assessment using a toxicity-exposure ratio, except that Annex VI to Directive 91/414/EEC stipulates the use of a safety factor of 10 whereas OECD (1996) recognises the potential need for a safety factor but does not recommend a specific value for it. A detailed scientific analysis would be required to assess whether the current EU safety factor of 10 takes appropriate account of all relevant issues, including those raised by the notifier in the case of pirimicarb (acuteness of exposure and the rate of ADME) and the substantial variation in toxicity between species. Two possible approaches to such an assessment have previously been suggested in an Opinion by the former Scientific Committee on Plants (SCP, 2002). The PPR Panel concludes that such an assessment would require substantial further work that is beyond the scope of this Opinion, and recommends that consideration be given to pursuing it as a separate initiative. In the meantime, the PPR Panel suggests that issues of the type raised by the notifier should be assessed case by case, as part of a refined, higher tier assessment. 2 Assessment question 2: assessment of the acute risk to birds from the use of pirimicarb 2.1 INTRODUCTION TOXICITY OF CARBAMATES: INHIBITION OF ACETYLCHOLINESTERASE (ACHE) The toxic effects of carbamates are due to inhibition of the enzyme acetylcholinesterase (AChE) which causes rapid accumulation of acetylcholine (ACh) in neuromuscular junctions and certain neural synapses. The excess of ACh 19 results in overstimulation of nicotinic and muscarinic receptors of autonomic organs and skeletal muscles, and in the central nervous system. Signs and symptoms of the cholinergic syndrome can be explained by considering the location of nicotinic and muscarinic ACh receptors (Taylor, 2001). On the basis of information obtained mainly with organophosphates, it has been shown that initial clinical signs of the cholinergic syndrome occur with at least 50% AChE 20 inhibition, whereas death is associated with more than 90% inhibition (Karalliedde et al., 2001). It should be noted that the dose-response relationship for AChE inhibition is log-linear. This means that 90% inhibition occurs at about 4 times the dose (concentration) required for 50% inhibition. 19 ACh: acetylcholine 20 AChE: acetylcholinesterase 6 of 21

7 2.1.2 CHARACTERISTICS OF INHIBITION OF ACHE BY CARBAMATES The mechanism of AChE inhibition by carbamates closely resembles the catalytic hydrolysis of ACh. Similar to the reaction with substrate, the carbamate forms a Michaelis-type complex with the enzyme. Then, the serine of the catalytic site reacts with the carbonyl group of the carbamate to form a carbamoylated AChE. This causes the inactivation (inhibition) of the enzyme. The inactivation of the enzyme is kinetically described by the bimolecular rate constant of inhibition (ki) which gives a measure of the inhibitory potency of the compound. From this constant, the IC50 (the concentration causing 50% inhibition at given conditions) can be calculated. Enzymatic activity is restored following displacement of the carbamoylated group with water. The kinetic of this process has been calculated for several compounds and depends on the carbamyl residue: e.g. for dimethylcarbamoylated AChE (such as that deriving after interaction with pirimicarb) the half-life of hydrolytic reactivation has been calculated to be minutes, irrespective of the dimethylcarbate that reacted with AChE (the half-life of the monomethylcarbamoylated AChE is somewhat lower). Consequently, complete recovery of AChE after inhibition by pirimicarb may occur within 3-4 hours. Because of the relatively rapid reactivation of carbamoylated AChE, carbamates are commonly, but erroneously, considered reversible inhibitors. In fact, a truly reversible inhibitor does not bind covalently to the enzymes and dissociates unchanged. On the contrary, carbamates are chemically altered when they inhibit AChE and reactivation of AChE entails hydrolysis of the carbamate which is consequently inactivated. This relatively rapid reactivation explains the methodological difficulties in the determination of AChE inhibition after treatment with carbamates. In fact, among others, timing of sampling, time of assay and dilution of the sample may be causes of underestimation of true AChE inhibition because they allow reactivation. Consequently, apparent discrepancies may occur between cholinergic signs and lack of or little measured AChE inhibition (Karalliedde et al., 2001) 2.2 TOXICOLOGICAL DATA DATA ON BIRDS: GAVAGE AND DIETARY EXPOSURES Pirimicarb toxicity data are available for bobwhite quail and mallard ducks (gavage, dietary and reproduction studies). Data on absorption, distribution, metabolism and excretion (ADME) are not available for birds BOBWHITE QUAIL Gavage administration of 45 mg/kg bw of pirimicarb to bobwhite quail (n=5 per group per sex) resulted in 100% mortality at 2 hours. At 19 mg/kg bw there were 3 deaths within 2 hours and survivors recovered from clinical signs within 24 hours, mostly within 6 hours (no observation between 6 and 24 hours). In males of this group food consumption was reduced by 30-40% during 4 days after dosing. No mortality was observed at 8 mg/kg bw and clinical signs were observed in some animals, these animals recovered by 6 hours. No effect was observed in food consumption. No clinical signs were observed at 3.3 mg/kg bw. The LD50 was calculated to be 20.9 ( ) mg/kg bw (van Dreumel and Leopold, 1996a). The dietary concentration of 2600 or 5200 ppm pirimicarb for 5 days caused 90% mortality in bobwhite quails, most deaths occurring on day 1. It is reported that animals became subdued within 2 hours after pirimicarb was offered in the diet but no mortalities were observed. No information on time to death or feeding pattern is available regarding the following 16 hours when animals were found dead. Food consumption was reduced by 30-50% in survivors during treatment (particularly days 2-5) and increased over control on day 6 onwards. At 1300 ppm slight clinical signs (animals were subdued ) were observed (time of onset not available) with 7 of 21

8 slight reduction in food consumption. The LC50 was calculated to be 1805 ppm ( ). No effects were observed at 650 ppm (corresponding to about 130 mg/kg bw). At 1300 and 2600 ppm the average daily dose is estimated to be in the range of 280 and 500 mg/kg bw, respectively, and at the LC50 it corresponds to an LD50 of about 370 mg/kg bw (Johnson and Dawe, 1997a). In a reproduction study conducted in bobwhite quail, no clinical signs (except reduced food consumption and associated reduced body weight) were observed at the top dose of 750 ppm (corresponding to about 64 mg/kg bw per day). At 300 ppm (corresponding to about 27 mg/kg bw per day) and below no effects were found on body weight and food consumption (Rodgers et al., 1997b) MALLARD DUCKS Gavage administration of 80 mg/kg bw of pirimicarb to mallard ducks (n=5 per sex) resulted in 100% mortality at 1 hour. At 25 mg/kg bw there were 3 deaths on day 1 (between 2 and 5 hours after treatment) and vomiting was observed soon after dosing in the 4 male survivors and in 4 female (2 that died and 2 survivors). Clinical signs occurred in some survivors, that recovered by 7 hours. No relationship was found between vomiting and mortality or (lack of) clinical signs. Reduced food intake was also found on days 1 (both sexes) and 2 (females) at this dose only. Neither mortality nor clinical signs were observed at 8 mg/kg bw except emesis in 3 males and 2 females soon after dosing. No clinical signs were observed at 2.5 mg/kg bw. The LD50 was calculated to be 28.5 ( ) mg/kg bw. However, given the occurrence of vomiting that may have significantly reduced the dose actually absorbed by the birds, the true LD50 might be lower than Since at 8 mg/kg bw no clinical sign was observed in the 3/3 males and 2/2 females that did not vomit, the LD50 value is probably higher than 8 mg/kg bw (van Dreumel and Leopold, 1996b). The dietary concentration of 2600 or 5200 ppm pirimicarb for 5 days caused 40-50% mortality in mallard ducks, deaths occurring on day 3 to 6 after start of dosing. Clinical signs (animals subdued and unsteady) were observed before day 3 until day 6 or earlier. Food consumption was reduced by about 30% at 650 and 1300 ppm and by about 70% or more at 2600 and 5200 during treatment. However, it should be noted that the reduction was more evident in the first two days of treatment; food consumption from day 3 was lower than control but significantly higher than on days 1-2. This might explain the appearance of clinical signs and mortality in the two top-dose groups from day 3 onwards. Slight reduction in food consumption was also observed at 6 to 8 days in the 2600 and 5200 ppm. The LC50 was calculated to be 5461 ppm ( ). At 1300, 2600 and 5200 ppm the average daily dose is estimated to be in the range of 360, 390 and 440 mg/kg bw, respectively, and at the LC50 corresponds to an LD50 of about 460 mg/kg bw (Johnson and Dawe, 1997b). In a reproduction study conducted in mallard ducks, no clinical signs (except reduced food consumption and associated reduced body weight during the first two weeks) were observed at the top dose of 750 ppm (corresponding to about 170 mg/kg bw per day). At 300 ppm (corresponding to about 60 mg/kg bw per day) and below no effect was found on body weight and food consumption (Rodgers et al., 1997a) CONCLUSIONS ON DATA ON BIRDS The clinical effects of pirimicarb occur rapidly and are reversible within a few hours; 8 of 21

9 Dietary concentrations of 750 ppm in bobwhite quail and in mallard ducks (corresponding to about 64 and 170 mg/kg bw, respectively) caused no clinical signs; Dietary concentrations of 1300 ppm (corresponding to about 280 mg/kg bw) caused mild clinical signs in bobwhite quail only; Food intake was reduced at 750 ppm and much more so at 1300 ppm and this is suggestive of avoidance; Dietary concentrations of 2600 ppm (corresponding to an acute dose of about 500 mg/kg bw) or more caused mortality within 1 day in bobwhite quail and on days 3-6 in mallard ducks. In the latter species, the lack of clinical signs on days 1-2 is likely explained by the very low food intake during those days as compared to day 3 onward. In bobwhite quail, the LC50 was estimated to be about 1805 ppm (corresponding to 370 mg/kg bw); Mortality was observed at much lower doses when pirimicarb was administered by gavage (LD50 about 20 mg/kg bw) SUPPORTING INFORMATION In the following, data on toxicity and ADME of pirimicarb in mammals are discussed to support the findings and conclusions on birds DATA ON MAMMALS ADME studies showed that absorption and elimination of pirimicarb was rapid in rats. In fact, following gavage administration of 1 mg/kg radiolabelled pirimicarb, almost 70% of the radioactivity was recovered from either urine (50-64%) or cage wash (2-6%) within 6 hours, and 83-89% within 24 hours (DAR Vol. 3, B6, pp ). In the acute neurotoxicity study in rats given pirimicarb by gavage, animals were observed at the time of peak for clinical cholinergic signs that was reported to be about 3 hours after dosing. The animals recovered by the following day (no indication of the specific time of recovery). Some deaths were observed at 110 mg/kg bw and 1 at 40 mg/kg bw. Cholinergic signs were also observed in all animals given 110 mg/kg bw and in 3/9 females at 40 mg/kg bw. The NOAEL (no observed adverse effect level) was 10 mg/kg bw (Horner, 1996a). In the 90-day neurotoxicity dietary study in rats, dose levels of 250 or 1000 ppm produced treatment-related effects on growth, with associated reductions in food consumption and/or food utilisation. These dietary concentrations corresponded to average daily intakes of 19.2 (range ) and 77.1 (range ) mg/kg bw, respectively. However, neither typical cholinergic signs, nor alteration in the Functional Observation Battery or motor activity were observed. The NOAEL 21 was 75 ppm, corresponding to an average daily intake of 5.6 mg/kg bw (range ) (Horner, 1996b). In the 1-year study in dogs given pirimicarb in capsules (25 or 35 mg/kg bw), onset of cholinergic symptoms was 2-4 hours after dosing and recovery 3-5 hours after dosing (Horner, 1998). In a published study on pregnant rats, a single dose of pirimicarb was administered by gavage on day 18 of pregnancy. The dose of 50 mg/kg caused 100% maternal mortality and maternal brain AChE was 80% inhibited when determined 15 minutes after dosing. The inhibition of AChE 21 NOAEL: no observed adverse effect level 9 of 21

10 in maternal liver and brain caused by 20 mg/kg was significant at 1 hour after dosing (33-62%) and showed substantial recovery at 5 hours after dosing (8-24%) (Cambon et al., 1979) CONCLUSIONS ON DATA ON MAMMALS Absorption, distribution, metabolism and excretion of pirimicarb are rapid; Animals recover from acute cholinergic signs as well as from AChE inhibition rapidly (few hours); this is consistent with the in vitro data on AChE reactivation; Cholinergic signs occur after a single gavage dose of 40 mg/kg bw, i.e., lower than the dose causing no such effect when administered via the diet ( mg/kg bw). 2.3 FEED INTAKE, AVOIDANCE RESPONSE, RATE OF ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION, AND REVERSIBILITY OF EFFECTS IN ACUTE RISK ASSESSMENT FOR BIRDS The studies reviewed in the preceding sections show: ADME and reversibility of effects are potentially important; Reduced food consumption in all birds studies, potentially contributing to reducing exposure; Cholinergic symptoms, recorded in birds about 1h and rats 15 minutes after exposure. The assessment of exposure and risk needs to take account of these factors and also the feeding rate of small birds eating small insects, which the notifier argued would be too slow to reach a lethal dose in one day. All these factors can be considered by extending an approach for assessing avoidance, which the PPR Panel proposed in an earlier opinion (EFSA, 2004). The following sections first set out the general approach and then apply it to pirimicarb GENERAL APPROACH The approach is developed in three stages: first considering the influence of avoidance (reduced food consumption) on acute exposure and risk, then adding the effect of ADME and recovery from toxicity, and finally some additional calculations that are needed to check that the feeding pattern implied by the assessment is realistic. The PPR Panel (EFSA, 2004) discussed the avoidance response in detail. For acutely toxic pesticides such as the organophosphates and carbamates there is extensive evidence that the main effect on food consumption does not occur from the start of exposure, but occurs at the point where the ingested dose reaches a level which causes cessation of feeding (the avoidance threshold). This threshold is below the lethal dose and might therefore be expected to prevent the animal from ever reaching the lethal dose. However, there is a delay between the time when the dose reaches the threshold and the actual cessation of feeding, due to the time taken for the ingested pesticide to be absorbed from the gut and reach target organs. Therefore it may be possible for the animal to reach a lethal dose, if the additional food consumption during the delay period takes the total dose above the lethal level. Expressing this in mathematical form, the total acute dose at the time when the bird stops feeding is given by: Acute Dose = AVT ( AVD FPM C) + bw Equation 1. where: 10 of 21

11 AVT = avoidance threshold dose (mg/kg bw) AVD = avoidance delay time (minutes) from reaching AVT to cessation of feeding FPM = feeding rate per minute (kg/min) C = concentration of pesticide in food (mg/kg) bw = body weight (kg) If the acute dose as estimated from equation 1 exceeds the LD50, then mortality in excess of 50% is expected. Alternatively, the acute dose can be compared to other endpoints (e.g. LD10 22 or a NOEL 23 ) if it is desired to assess the occurrence of lower mortality rates or non-lethal effects. Equation 1 however does not allow for the effect of ADME and the reversibility of effects, which are significant in the case of pirimicarb. The anticholinesterase mode of action together with clinical evidence from the available avian and mammalian studies indicate that the ADME processes are likely to be first order, and can therefore be characterised by an exponential decay with a rate constant representing their combined effect. To take account of these processes, each part of the total dose in equation 1 (the avoidance threshold dose, and the additional dose ingested during the avoidance delay period) is reduced by an amount that depends on the rate of the ADME processes and the duration of the avoidance delay period. This can be incorporated into the assessment by adapting Equation 1 to produce Equation 2 as follows 24 : Acute Dose = AVT e k AVD + FPM C) (1 e ( bw k) ( k AVD ) Equation 2. where the rate constant k = ln (2) / (half life of combined ADME processes) 25. It is important to consider what data are available to estimate the parameters in equation 2. Although standard toxicological and ADME studies are not designed to measure AVT and AVD, they may contain sufficient information for the assessor to estimate approximate upper and lower bounds, as will be illustrated below for the case of pirimicarb. C and bw may be estimated in the same way as in the standard avian exposure assessment (SANCO, 2002a) 26. FPM however is different from the food intake rate (FIR) used in the standard assessment: FIR is the total daily food requirement, whereas equation 1 and 2 relate to just the first bout of feeding on contaminated food and so FPM should be the feeding rate in kilograms per minute during that first bout. Data on feeding rates in the wild are available for a few species; although these studies very rarely relate to contaminated food and often not to the food type of interest, they may be sufficient for the assessor to estimate approximate upper and lower bounds for FPM in the scenario of interest. Again this is illustrated for the case of pirimicarb below. Standard ADME studies may provide an estimate of k but are generally conducted with mammals, and extrapolation of k from mammals to birds will be uncertain. For some pesticides a chicken metabolism study is conducted, but the relationship between ADME processes in chicken and other bird species is also uncertain. The PPR Panel therefore identified two further 22 LD10: Lethal dose; the dose at which 10 % of the test organisms die 23 NOEL: no observed effect level 24 See Annex 1 for the derivation of equation Note: ln(2) denotes the natural logarithm of Note that equation 1 excludes the factors PT (proportion of food obtained in treated area) and PD (proportion of food type in diet), which are part of the standard avian exposure assessment (SANCO, 2002a). This is because the key period in determining whether avoidance will fail to prevent mortality is the initial bout of feeding on first encountering contaminated food. Although this first bout might include a mixture of food types, it will often contain a single food type obtained entirely within the treated area (thus PD=1 and PT=1 for the duration of the bout) of 21

12 approaches for obtaining approximate estimates of k, using information from the standard avian toxicity studies. The first approach makes use of information from the acute avian LD50 (gavage) study. It is assumed that during the acute recovery time (ART) taken for disappearance of clinical signs in animals at the lowest observed effect level (LOEL), ADME processes reduce their effective dose to the no observed effect level (NOEL). On these assumptions, k may be estimated as: k = NOEL ln LOEL ART Equation 3. This estimate is very uncertain and could either over- or under-estimate k, because the observed NOEL 27 and LOEL 28 will be respectively below and above the observed values (therefore overestimating the dose eliminated), whereas the true recovery time will be less than the observed time. The second approach for estimating k in birds uses the NOEL for clinical effects in dietary studies (either LC50 or reproductive), the LOEL for clinical effects in the acute oral LD50 study in the same species, and the duration of the exposure period (DEP) in the dietary study. It is assumed that birds in the dietary study have survived an average daily dose equal to the NOEL for a period DEP without reaching an internal dose equivalent to the acute LOEL. On these assumptions, the rate constant k can be estimated via the following equation: LOEL = NOEL k (1 e k DEP ) Equation 4. Unfortunately equation 4 cannot be rearranged to give a precise solution for k, so it is necessary to find k by iterative calculation. This is done by calculating equation 4 repeatedly to find the value of k that gives the observed value for LOEL. An example of this iterative calculation is shown later for pirimicarb in Table 1. Equation 4 makes the simplifying assumption that food intake is spread evenly over 24 hours per day, and gives only an uncertain estimate of k. However, it should be conservative for the species tested, because the true NOEL and LOEL will be respectively above and below the observed values. It should be noted that if k is high enough, then the rate of the ADME processes at the LD50 dose (LD50 x k) may exceed the rate of dose ingestion (FPM x C/bw). In such cases it is not possible for the animal to reach the LD50, even if there is no avoidance response. It is useful to check for this, as it may avoid the need to estimate the other parameters in equation 2 (AVD, AVT). If it appears that the animal may achieve a lethal dose despite any effects of ADME and avoidance, it is important to check the realism of the total food ingestion required to reach the lethal dose (taking account of the effects of ADME). The total time taken from the start of feeding until the animal reaches the LD50 can be estimated as follows: 27 NOEL: no observed effect level 28 LOEL: lowest observed effect level 12 of 21

13 Time to reach LD 1 = ln k 50 1 LD50 k bw FPM C Equation 5. The result of equation 5 can then be multiplied by the feeding rate to estimate the total food intake required to reach the LD50. If field evidence shows that birds are unlikely to feed continuously for the period estimated by equation 5, or if the amount of food consumed in this time would substantially exceed the expected daily intake, then it might be concluded that such an exposure was unlikely to be achieved in the field. All of the parameters in equations 1-5 are uncertain, to varying extents, and it is important to explore the impact of this uncertainty on the risk assessment. A simple way to do this is to repeat the calculations several times using different assumptions (e.g. worst-case and more favourable) for each parameter. If the worst-case results are sufficiently below a lethal exposure, then it might be concluded that the acute risk to birds was acceptable 29. If even the most favourable assumptions indicate that lethal exposures will occur frequently, then it might be concluded that the risk is unacceptable. In intermediate cases, where worst-case results suggest mortality but favourable assumptions lead to exposures well below the LD50, it may be worth seeking extra data on the parameters in equation 2 to assess the risk with more certainty ASSESSMENT FOR PIRIMICARB CHOICE OF SPECIES AND ESTIMATION OF FEEDING RATE In the case of pirimicarb, the PPR Panel first considered which wild bird species to conduct the calculations for, taking account of their use of relevant habitats (cereal crops), their diets (inclusion of small insects) and the availability of information on feeding rates. The RMS assessment mentions blue tits (Parus caeruleus) as an example of a small insectivorous bird, but the PPR Panel was unable to find any data on rates of feeding by this species in the field. Davies (1977) estimated that the energy intake rate of yellow wagtails (Motacilla flava) feeding on insects at dung pats was 285 J/min, which is equivalent to approximately g/min (assuming energy and moisture contents quoted in Appendix I of SANCO 2002a). Davies (1977) found lower intake rates for yellow wagtails feeding on insects at pools: 196 J/min or g/min. Yellow wagtails are found in cereal fields, and it is conceivable they could reach similar feeding rates there if insects were abundant. They have also been recorded feeding on dead insects when present in high densities (Dittberner, 1984), which might occur after application of pirimicarb. Green (1978) provides detailed information on rates of feeding by skylarks (Alauda arvensis), which are found in cereal fields at higher densities than yellow wagtails (Mason & Macdonald, 2000). Intake rates estimated from Green s (1978) data for skylark range higher and lower than those quoted above for wagtail, but relate to feeding on spilt or exposed grain on cereal sowings and stubbles (0.18 g/min) or weed seeds on ploughed fields (0.019 g/min) rather than insects. It is conceivable that skylarks might consume insects at the same rate as cereal grain, if they were abundant enough, so the high and low estimates for skylarks will be used in the favourable and worst-case assessments respectively (if the results using the high estimate indicated a potentially unacceptable risk, more detailed consideration might then be given to this issue). The body weight (bw) of skylarks is assumed to be 37g (average value from Dunning, 1992). 29 Note that judgements of acceptability, including whether to base the assessment on the LD50 or a lower endpoint such as LD10 or NOEL, involve policy considerations that are part of risk management and outside the remit of EFSA of 21

14 ESTIMATION (ASSUMPTION) OF LD50 The toxicity of a pesticide usually varies by one or more order of magnitude between different species of birds (Luttik and Aldenberg, 1997), so it is necessary to allow for the possibility that birds exposed in the field may be more (or less) sensitive than the species tested in the laboratory. In the first tier assessment this is considered to be accounted for within the uncertainty factor of 10 that is used to evaluate the toxicity-exposure ratio, but in higher tier assessments statistical approaches to species differences in toxicity may be considered (SANCO 2002a). The PPR Panel therefore conducted its calculations assuming that skylark LD50 is 3.67 mg/kg bw, which is the median estimate of the HD5 (fifth percentile of the distribution of LD50s between species, i.e. 5% of species are expected to have LD50s below the HD5 30 ) and is obtained by dividing the bobwhite quail LD50 by 5.7 (method of Luttik and Aldenberg, 1997) ESTIMATION OF AVOIDANCE THRESHOLD DOSE (AVT) The PPR Panel estimated the avoidance threshold dose (AVT) by examining the dose level at which food consumption is reduced in the acute dietary study, rather than the dietary studies where estimating AVT is difficult due to the effects of the ADME processes. In the acute oral study with bobwhite quail, the NOEL and LOEL for reduced food consumption were 8 and 19 mg/kg bw respectively, suggesting that AVT for bobwhite quail is between these two doses, i.e. between 38 and 91% of the LD50 for the same species (20.9 mg/kg bw). The PPR Panel assumed that AVT will be proportionately reduced or increased in other species with lower or higher LD50s. Therefore, in its worst case calculation the PPR Panel assumed an AVT of 3.33 mg/kg bw (91% of the HD5 32 ), and in its favourable calculation, an AVT of 1.39 mg/kg bw (38% of the HD5) ESTIMATION OF AVOIDANCE DELAY TIME (AVD) The PPR Panel estimated the avoidance delay time AVD from the time to first overt symptoms in acute toxicity studies, assuming that animals suffering overt symptoms are likely also to stop feeding. The earliest recorded symptoms in acute studies with birds occurred one hour after dosing of mallards, and the earliest for mammals were after 15 minutes in a study with pregnant rats (extrapolation to birds is uncertain, but evidence from other pesticides makes it plausible e.g. EFSA, 2004). The PPR Panel therefore assumed AVT=60 minutes in its worst-case calculation, and 15 minutes in its favourable calculation ESTIMATION OF CONCENTRATION OF PIRIMICARB ON SMALL INSECTS The PPR Panel made the same assumption as the Rapporteur Member State (RMS) regarding the concentration of pirimicarb on small insects (C = 6.09 mg/kg) ESTIMATION OF HALF-LIFE FOR ELIMINATION OF PIRIMICARB. The PPR Panel considered four alternative approaches to estimating the half-life for elimination of pirimicarb. First, as noted in section 2.1.2, the half-life of hydrolytic reactivation for dimethylcarbamoylated AChE (such as that deriving after interaction with pirimicarb) has been calculated to be minutes in mammals. Although this represents only one of the constituent processes and may not be the rate-limiting step, the half-life for overall clearance of 30 The PPR Panel does not wish to suggest a general precedent by using the HD5; more or less protective assumptions could be substituted if preferred. 31 The method is applied here as if only a single LD50 was available, because the mallard LD50 for pirimicarb is uncertain due to regurgitation. 32 HD5: fifth percentile of the distribution of LD50s between species 33 Note that this is based on a residue per unit dose (RUD) of 29, rather than the more conservative RUD of 52 specified by SANCO (2002a, p. 15) of 21

15 pirimicarb may not be much larger. The second approach is based on the observation that almost 70% of radioactivity was recovered within 6 hours in the mammalian metabolism study. Applying equation 3 with ART 34 = 360 minutes and replacing NOEL/LOEL with 0.3 (the proportion of dose remaining) leads to an estimated k of mins -1 and half-life of 207 minutes. The third approach applied equation 3 to the acute LD50 study with bobwhite quail, giving an estimated k of mins -1 and half-life of 282 minutes. The fourth approach used equation 4 and the LOEL from the LD50 study with bobwhite 35. When this was applied to the bobwhite and mallard LC50 studies and the bobwhite and mallard reproduction studies it gave estimated k values corresponding to half-lives of 62, 23, 125 and 47 minutes respectively. The iterative calculation for obtaining these results using equation 4 is illustrated in Table 1. The PPR Panel decided to use half-lives of 23 and 282 minutes respectively in its favourable and worst-case calculations. These reflect the high uncertainty indicated by the results of the four approaches to estimating the half-life, although the worst-case estimate is probably very conservative. Table 1. Iterative calculation of Equation 4 to estimate the ADME rate constant k given NOEL = 64 mg/kg bw/day and duration of the exposure period (DEP) = 20 weeks (from the reproduction study with bobwhite quail). A half life of 125 minutes gives a dose corresponding to the observed acute bobwhite LOEL of 8 mg/kg bw. half life (minutes) k LOEL ESTIMATION OF DOSE AND CONSIDERATION OF UNCERTAINTIES The PPR Panel made two sets of calculations using equations 1-5: one set with the worst-case assumptions outlined above, and the other set with the more favourable assumptions. If the favourable assumptions were true, there would be no possibility of a skylark reaching the LD50 even without avoidance, because at that dose level the rate of metabolism/elimination (LD50 x k = 0.11 mg/kg/min) would greatly exceed the rate of dose ingestion (FPM x C/bw = mg/kg/min). In the case of pirimicarb, the ADME processes are known to be important so the worst-case assessment is based on equation 2 rather than equation 1. With the worst case half life of 282 minutes, equation 2 shows the net dose at the onset of avoidance would be 4.6 mg/kg bw, i.e times the assumed LD50. Under these assumptions, equation 5 shows that the skylark would have to feed at the worst case feeding rate for 143 minutes, during which it would ingest 26g of small insects (equal to 71% of its body weight, and 102% of the amount of insects needed to meet its estimated daily energy requirement), which seems fairly unlikely. The net dose for yellow wagtails under worst-case assumptions was 1.02 times the LD50. However, realistic relaxations in only one or two of the conservative assumptions in the assessment (AVT, AVD, FPM, k, LD50) would be sufficient to bring the net doses below the LD50. It therefore 34 ART: acute recovery time 35 The mallard LD50 study was not used here due to uncertainty caused by emesis of 21