A 1-year toxicity study in dogs is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides

Contents

Abstract �� 11� Introduction �� 12� Individual reviews of dog studies and their conclusions �� 23� Synthesis of the data �� 7 3�1� Conceptual approaches/differences �� 7 3�2� Size of the entire dataset �� 8 3�3� How were compounds judged in the reviews? �� 104� Conclusions/recommendations �� 11Appendix �� 11Acknowledgements �� 15Declaration of interest �� 15References �� 15

(Received 06 June 2009; revised 31 August 2009; accepted 31 August 2009)

ISSN 1040-8444 print/ISSN 1547-6898 online © 2010 Informa UK LtdDOI: 10.3109/10408440903300098 http://www.informahealthcare.com/txc

R E V I E W A R T I C L E

A 1-year toxicity study in dogs is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides

Werner Kobel1, Ivana Fegert2, Richard Billington3, Richard Lewis4, Karin Bentley5, Werner Bomann6, Phil Botham4, Bernhard Stahl6, Bennard van Ravenzwaay2, and Horst Spielmann7

1ToxAdvice GmbH, Reinach BL, Switzerland, 2BASF, Ludwigshafen, Germany, 3Dow AgroSciences, Abingdon, United Kingdom, 4Syngenta Crop Protection, Jealotts Hill, United Kingdom, 5Du Pont de Nemours, Newark, Delaware, USA, 6Bayer CropScience, Sophia Antipolis, France, and 7Freie Universität Berlin, Germany, & BfR (Federal Institute for Risk Assessment), Berlin, Germany

AbstractA review of publications on pesticides assessing the need for 1-year toxicity studies in dogs was performed. Four key peer-reviewed papers with different approaches investigated the value of a 1-year dog study in addition to a 3-month study. Despite different databases and approaches, each concluded with the recommendation to limit the testing of pesticides in dogs to a duration of 3 months. The combined weight of evidence presented in this review reinforces these independent conclusions. Therefore, the routine inclusion of a 1-year dog study as a mandated regulatory requirement for the safety assessment of pesticides is no longer justifiable and a globally harmonized approach should be taken to match the latest legislation of the European Union and the US EPA.

Keywords: Dogs; pesticides; repeat-dose toxicity studies; study duration

Critical Reviews in Toxicology, 2010; 40(1): 1–15Critical Reviews in Toxicology

2010

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06 June 2009

31 August 2009

31 August 2009

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© 2010 Informa UK Ltd

10�3109/10408440903300098

Address for Correpondence: Dr. Werner Kobel, Dr. med. vet., FVH, ERT, ToxAdvice GmbH, Christoph Merian-Ring 11, CH-4153, Reinach BL, Switzerland. E-mail: [email protected]

TXC

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1. Introduction

Safety for users and consumers is a key element in the development of crop protection products and their active ingredients� The toxicology database required for the regis-tration of a pesticide has been developed since the 1950s�

Repeat-dose studies with rodents and nonrodents are a core element, and for more than three decades it was an established standard to perform subchronic and chronic oral studies in rats and dogs in order to evaluate systemic toxicity in accordance with international (OECD, EU) and

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2 W. Kobel et al.

national test guidelines (US EPA, JMAFF, and many others)� Subchronic studies normally last for 3 months, whereas chronic studies should last for at least 1 year� For chemi-cals, including pesticides, the primary objective of these studies is to identify the dose at which no adverse effects occur—the no observed adverse effect level (NOAEL)—which is then used to set a safe reference dose for humans and enable a risk assessment to be performed� Effects at higher dose levels may be used for other purposes, such as classification and labelling, but these do not tend to be the primary objective of these types of toxicity studies, espe-cially in dogs� In order to avoid diversion of the discussion into areas for which the needs are different from those of pesticides, such as pharmaceuticals, this review is limited to those publications addressing pesticides� For complete-ness and where needed, reference is made to pharmaceu-ticals in order to show where differences may exist, and are justified�

Testing and study guideline requirements have increased over the last 20 years, calling for more studies and/or more study parameters, in an attempt to provide more compre-hensive safety databases� On the other hand, this increase in requirements has also led to animal welfare concerns about the justified use of animals, in particular the use of dogs� Although initially discussed within pharmaceutical circles (Lumley et al�, 1985, 1992, 1993), there has also been an extensive discussion on what constitutes an “adequate” safety dossier for pesticides in general and the particular role of the dog studies (Gerbracht & Spielmann, 1998; Spielmann & Gerbracht, 2001; Box & Spielmann, 2005; Baetcke et el�, 2005; Doe et al�, 2006; US EPA, 2006)� The need for dog stud-ies has been affirmed unanimously by all authors� They all confirm the necessity of the dog as a second (or nonrodent) species, which is consistent with current worldwide regula-tory requirements for the safety assessment of pesticides as well as the regulatory requirements for the preclinical safety assessment of pharmaceutical compounds (Broadhead et al�, 2000; Smith, 2002)�

During the past three decades, it has been necessary to perform both a 3-month and a 1-year toxicity study in dogs in support of the global registration of pesticides� The large number of studies performed during this period has pro-vided an extensive database that has been used to address the relational value of these two studies of differing duration (Spielmann & Gerbracht, 2001; Baetcke et al�, 2005)� Further reviews by Box and Spielmann (2005), Doe et al� (2006), and US EPA (2006) consider this particular point in the wider context of varying approaches to agrochemical safety assessment�

The regulatory practice is not uniform around the globe with regards to the requirement of a 3-month and a 12-month toxicity study in dogs� The revised Directive 91/414 (Revision to Annexes II and III to Directive 91/414/EEC, Sanco/10482/2006 rev�11) in Europe now limits dog studies to 90 days and does not require a 12-month study� This change is consistent with a recent decision by the US EPA (CFR 158�500, 26� Oct 2007, p 60976, for further details

see footnote 1) to no longer require a 12-month study in dogs� Although no official notification is currently available from PMRA Canada and APVMA Australia, personal com-munications have indicated that these countries will take a similar position� Other countries such as Brazil, Argentina, and Japan have yet to consider their position�

The purpose of this review is to critically evaluate the weight of evidence relating to the need for a subchronic (3-month) and a chronic (12-month) toxicity study in dogs and to determine impact that each has on the safety evalua-tion of pesticide products�

2. Individual reviews of dog studies and their conclusions

A thorough review on original data on file at the German BgVV (Federal Institute of Health Protection of Consumers and Veterinary Medicine), now Bundesamt für Risikobewertung (BfR), was performed by Gerbracht and Spielmann (1998)�2 Data from 232 pesticides (i�e�, acaricides, fungicides, growth regulators and hormones, herbicides, insecticides, mollusci-cides, nematicides, rodenticides, synergists for insecticides) generated between 1953 and 1995 were examined� The databases of 216 pesticides were found to contain suitable short-term subacute (4 weeks), subchronic (13 weeks), and/or long-term chronic (52 weeks or longer) studies in dogs, rats, and mice, and were examined by evaluation of the rel-evance of the no observed effect levels (NOELs) for the safety assessment for each chemical�3 The authors concluded that studies in dogs were necessary as a second species in regula-tory testing because this species proved more sensitive than rats or mice in a number of cases when the NOELs and low observed effect levels (LOELs) were compared� To maintain confidentiality, the data owners (submitters) of the stud-ies requested that data for substances in this review were coded� This limited the comparisons that could be made in this paper with individual compound data in other datasets (Baetke et al�, 2005; Doe et al�, 2006)�

In a subsequent paper, Spielmann and Gerbracht (2001) performed an analysis of the database established for the SET/BfR study in the 1998 publication above� Of the 216 pesticides with suitable toxicity data, 172 had short-term and chronic dog studies� The aim of this publication was to assess whether chronic studies in dogs (52/104 weeks)

1A 1-year nonrodent study (i�e�, 1-year dog study) would be required if the Agency finds that a pesticide chemical is highly bioaccumulating and is eliminated so slowly that it does not achieve steady state or sufficient tissue concentrations to elicit an effect during a 90-day study� EPA would require the appropriate tier II metabolism and pharmacokinetic studies to evaluate more precisely bioavailability, half-life, and steady state to determine if a longer-duration dog toxicity study is needed�2 This study was sponsored by SET (Stiftung zur Förderung der Erforschung von Ersatz- und Ergänzungsmethoden zur Einschränkung von Tierversuchen), the German Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing and is referred to later on as the SET/BfR study�3 The duration of the chronic mouse studies has not been described, it is assumed to be 78 weeks�

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Review on 1-year dog studies for pesticides 3

provide essential additional specific information compared to subchronic (13 weeks) or subacute (4 weeks) studies in this species� Studies were initially ranked according to the LOELs (rank 1 lowest, rank 3 highest LOEL)� The subacute studies proved to be the most insensitive studies, as they had the highest rank values� No clear difference in sensitiv-ity was found between the rankings of the subchronic and chronic studies� The distribution of the ratios between the LOELs of the subchronic and chronic studies did not show a consistent distribution pattern for insecticides, herbicides, and fungicides� Organ toxicity was subsequently assessed by comparing shorter- versus longer-term studies, mostly (4- and) 13-week versus the 52/104-week studies� As a general rule, the same target organs were identified in both subchronic and chronic studies� Comparisons were made on an individual compound basis with a focus on new informa-tion found in chronic dog studies relative to effects observed in the corresponding subchronic or subacute studies� The incidences are listed in Table 1�

The authors included a fairly detailed evaluation of the potential value of the 26-week data but the sample size was relatively small (only 13 studies in total)� Of most interest in this publication was a comprehensive comparison of the findings in the 13-week and the 52/104-week studies, which also comprised the bulk of available data�

New findings in chronic studies (52/104 weeks) were categorized according to toxicological relevance, in terms of the adverse nature of the finding� If the new finding was judged to be not toxicologically significant, then the study was eliminated from further considerations� Findings were not considered to be relevant when

only some isolated nonspecific parameters were altered •(i�e�, only organ weight or enzymatic activity in blood

samples without a functional or histopathological correlate);higher doses were used in chronic studies;•affected organs observed in chronic studies in dogs •were also identified in rats or mice; orthe study author concluded that the findings were not •considered relevant for toxicity in humans�

The results are summarized in Table 2�For 68 of the 141 compounds, all salient effects in dogs

were identified within 13 weeks of treatment� For 73 com-pounds, new findings were seen in chronic studies, which had not been observed in subchronic studies� However, in 27 of these cases, the affected organs were also identified in rodent studies, and for 13 additional compounds, the new findings were seen only at higher doses not previously tested in the subchronic studies� For a further 18 compounds, substantial evidence for toxic side effects was missing, as histopathological findings did not correlate with the clinical symptoms reported in the studies� For 8 more compounds, the new findings were not of toxicological relevance in the opinion of the toxicologist who carried out the study� Thus, there were 7 out of 141 compounds (5�0%) for which relevant new toxicological findings in the chronic 52/104-week dog studies were not detected in 13-week studies� The number of these findings that occurred only after 104 weeks of expo-sure has not been specified� The authors did not identify in the result section which coded compounds eventually were judged to be among those with relevant toxicological effects� At some stage they used common names to discuss findings, though without reference to the code�

Dichlofluanid, pyrazophos, and rimsulfuron are among those supporting the need for a 12-month study, as they belong to the group of 7/141� The first two, together with trinexapac ethyl, fosetyl AI, iprodion, bromuconazole, dini-conazole, and fluazinam, belong to the group of 8/54 fungi-cides, which were commented in some more detail�

This limits the possibilities to compare the judgement on individual compounds with the assessments done in the other publications�

Table 1. Incidence of new findings in chronic dog studies not seen in subchronic or subacute studies (Spielmann & Gerbracht, 2001).

Fungicides Herbicides Insecticides Others Total

All data 30/55* 25/44 17/38 10/16 82/153

*No. of new findings /available study sets for comparison.

Table 2. Analysis of toxicity studies in dogs on herbicides, insecticides, fungicides, and other pesticides (52/l04-week versus l3-week studies).

Herbicides Insecticides Fungicides Other pesticides All

Total number (of compounds) 42 32 54 13 141

All salient effects in dogs identified within 13 weeks 18 20 25 5 68

New salient findings in dogs identified in chronic studies 24 12 29 8 73

New salient findings in chronic studies that were seen in studies in rats/mice 11 5 8 3 27

Higher doses in chronic studies than in 13 week studies 3 4 5 1 13

Toxicological effects without relevance a) 6 2 8 2 18

Symptoms without toxicological effect in toxicologist’s expert opinion b) 2 0 4 2 8

Relevant toxicological effects (seen in 52/104-week vs. 13-week dog studies) 2 1 4 0 7Note. From original publication (Spielmann & Gerbracht, 2001).aToxiclogical effects without relevance included effects on organ weights without a histopathological and/or functional correlate; single, isolated alterations in enzymatic activity without organ weight change or histopathological correlate; clinical effects on the CNS when the chemical was administered via capsule or stomach tube, or if isolated symptoms were diagnosed (only emesis, etc.); effects on hematopoietic system only if a single hematological parameter was affected without a histopathological correlate or if hemosiderosis was found without a hematological correlate; effects on gastrointestinal tract (e.g., diarrhoea or fluid feces) without a histopathological correlate.bThese symptoms were found neither in 13-week studies in dogs nor in studies in rats and mice. Following the expert opinion of the toxicologist, these symptoms were taken as having no toxicological relevance.

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4 W. Kobel et al.

On the basis of this analysis, the authors stated the follow-ing: “Our analysis of studies in dogs suggest that only in 7 of 141 cases was new relevant information provided by chronic 52 or 104 week studies, which was seen neither in subchronic stud-ies on dogs nor in chronic studies in rats. Our data prove that most of the organ-specific toxicity of pesticides can be identi-fied in 13-week studies in dogs and that all significant toxic effects are identified after 26 weeks of exposure. Thus safety testing of pesticides in dogs should be limited to subchronic (13 week) studies since an extension of the duration of the studies does not provide additional essential information.”

As part of the ILSI HESI Agricultural Chemical Safety Assessment (ACSA) technical committee, Doe et al� (2006) reviewed the current regulatory requirements with regards to the assessment of potential systemic toxicity testing� To support their evaluation, the SABRE (Safety Assessment By Refined Experimentation) database was developed� A limited number of substances were chosen from the major pesticide classes for which the toxicology databases had been evaluated by the Hazard Identification Assessment Review Committee (HIARC) of the Health Effects Division (HED), Office of Pesticide Programs (OPP), and US EPA� The study endpoints, namely no observed adverse effect levels (NOAELs), low observed adverse effect levels (LOAELs), reference doses (RfDs), and others, were carefully reviewed, taking into account further regulatory documents such as Registration Eligibility Documents (RED) or Data Evaluation Reports (DER)� Altogether, 65 compounds were entered into the SABRE database, of which 26 had 90-day and 1-year dog studies suitable for comparative assessments� Of these 26 substances, 15 had been included in the evaluation by Gerbracht and Spielmann in their first publication (1998)�4

The approach chosen was to compare the NOAELs of the subchronic and chronic dog studies� To be considered as dif-ferent, the levels had to be apart by a factor of 2 or higher� For 8 compounds, the 1-year study yielded a lower NOAEL� For three of the compounds (2,4-DB, iprodione, propachlor), the rat was more sensitive, thus the dog values were not considered to be relevant for deriving the reference doses� To assess the potential influence of dose setting, the dog LOAELs and the ratio between the NOAELs in subchronic and chronic studies in rats and dogs were also considered� For one compound (hexaconazole), the difference was within a factor of 3, thus close to 2, and this applied not only for the NOAELs but also for the LOAELs� For another compound (acetochlor) there were two 1-year dog stud-ies, of which the NOAEL of one was within the factor of 2 of the NOAEL of the subchronic study, whereas the factor for other was 5� Three compounds remained, namely butylate, tebuconazole, lactofen, 5 in which the 1-year dog NOAEL was judged to be lower than that of the 90-day study by more than a factor of 2 set as margin of concern� For lactofen and tebuconazole, the results were not due to the dose selection�

For tebuconazole and lactofen, the dog 90-day values were considerably higher than any other NOAELs� Butylate was found to be difficult to understand, because the 12-month dog NOAEL and LOAEL are unusually low when compared to the other NOAELs, thus was also considered to show a possible difference� The case of butylate is discussed in more detail below�

Doe and coauthors further examined the impact of the regulatory outcome if the 12-month dog study was not per-formed� In this endeavor, they identified the lowest NOAEL in the standard set of four systemic toxicity studies (90-day rat, 2-year rat, 90-day dog, 1-year dog) for each substance and compared the result with and without consideration of the 12-month dog study� They found that for only two com-pounds (acetochlor and butylate), the lowest NOAEL from the other three studies are more than twice the NOAEL for the 12-month dog study� As noted above, for acetochlor, there are confounding factors with its two 1-year dog stud-ies� The database for butylate again was found to be unusual (discussed later)�

In the publication, as an interim conclusion, it was stated: “The Task Force agreed that performing a 90 day study in the dog meets the objective concerning the relative sensitivity of the rat and the dog, while at the same time providing infor-mation that would be relevant for the 1 to 6 month human exposure period. “

In 2005, Box and Spielmann published an additional review that included a search of publicly available literature from 1974 to 2004� The authors reported that approximately 25 relevant publications were found, which reported nine subchronic and chronic dog studies for single pesticides� None of the studies described findings in the chronic studies that had not been observed in the subchronic studies�

It should be noted that the data of Doe et al� (2006) were considered� However, the comprehensive review by Baetcke et al� (2005) was not included�

Box and Spielmann (2005) suggested an approach consist-ent with current International Conference on Harmonisation (ICH) practice where dog studies of durations between 3 and 12 months can be conducted� The authors propose a general (generic) safety factor to be applied on the NOAEL of the chronic rat study for all cases in which the dog may appear more sensitive/susceptible than the rat in subchronic stud-ies� This approach may have its merits from the point of view of reducing the use of dogs, but does not do scientific jus-tice to compounds under investigation� As has been shown and concluded by Spielmann and Gerbracht (2001), Doe et al� (2006), and Baetcke et al� (2005), a chronic dog study is rarely of relevant influence in the derivation of the reference doses (e�g�, acceptable daily intake [ADI], acceptable opera-tor exposure level [AOEL], etc�) for a pesticide� A generic additional uncertainty factor if the dog appears to be more sensitive/susceptible in subchronic studies would reduce

4Benomyl, bifenthrin, (carboxin), chlorfenapyr, cyprodinil, diazinon, dimethoate, fosetyl, glufosinate, iprodione, MCPA, metolachlor, tebucona-zole, triallate, vinclozoline�

5Butylate and lactofen are not included in Spielmann and Gerbracht (1998) or Baetcke et al� (2005)� Tebuconazole is listed in Spielmann and Gerbracht (1998), but not mentioned otherwise�

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the reference dose for a number of compounds in an unjus-tified manner� A careful scientific analysis of the database is of much higher value for the identification of these poten-tially possible, but rare cases� Nevertheless, if the idea of a generic safety factor should be pursued as suggested by Box and Spielmann (2005) and assuming there are subchronic studies available for rats and dogs, then a more compound specific approach should be taken where a correction factor is based on the ratio between the NOAELs of the subchronic rat and dog studies, provided this ratio exceeds a value of 1�5 or 2, which are reasonable limits applied by Doe et al� (2006) and Baetcke et al� (2005) in their analyses�

Although there may be merits for such an approach purely from the animal welfare perspective, it would not be an approach based on science and data� It should also be kept in mind that there is a standard uncertainty value of 100 (10 × 10) applied when setting reference doses, which in the experience of several decades has proven its value (Renwick, 2000)�

Baetcke and coauthors of US EPA (2005) conducted a comparison of the results in dog studies with pesticides of 12/24 months’ duration to those of dog studies of shorter duration� This paper is not a standard peer-reviewed pub-lication� However, the review was submitted and discussed extensively at a meeting of the US EPA FIFRA Scientific Advisory Panel on May 5–6, 2005; thus it can be considered to have the status of a peer-reviewed publication�

The original database of this study comprised dossiers of 304 compounds, for which the US EPA OPPTS established chronic RfDs� Of these, 116 (38%) were based on dog stud-ies� For the other 62%, any effects in the dog occurred only at doses above those producing effects in other species and as a consequence, neither the 13-week nor the 1-year study contributed to the determination of the RfD� The subset of 116 (38%) compounds then was further analyzed to deter-mine whether the available data were sufficient to compare the results of dog studies of at least 1 year with studies of shorter duration� The following criteria were used:

Availability of a dog study with a duration of ≥1 year and •a dog study with a duration of 13 weeksAvailability of results from a dog study of ≥1 year’s dura-•tion that included clinical or haematology measure-ments at 13 weeks or less

Pesticides not yet registered or those with cancelled reg-istrations were not included� Additional information such as Data Evaluation Reports (DER) and reports of the EPA Hazard Identification, Assessment, and Review Committee (HIARC) were also taken into consideration�

There were 77 pesticides identified that satisfied the crite-ria above� Forty-two had studies of duration of 1 year or more and studies of 13 weeks or less� An additional 35 pesticides had no separate 13-week studies, but shorter-term/interim-term clinical chemistry or haematologic measurements were available� For most of these 35 pesticides, no interim histopathologic data were available� In these cases, the

comparisons had to be limited to the LOAEL of the clinical chemistry and haematology� For cholinesterase inhibitors, brain cholinesterase activity could also not be considered, as it cannot be measured intra vitam� The criterion to consider NOAELs or LOAELs as different was set at a factor of ≥1�5�

In a first step, the NOAELs and LOAELs were listed and the factors calculated� In the group of 42 compounds for which studies of different durations were available, 23 showed no difference greater than a factor of 1�5� In the group of 35 sub-stances where interim data were limited to in-life data, clini-cal chemistry and hematology, there were no differences found for any of the compounds�

The 19 (42 minus 23) compounds for which a difference was found were further scrutinized�6 In 11 cases, the analysis of the dose-effect data indicated that comparable NOAELs/LOAELs would have been identified if these pesticides were evaluated in well-conducted 13-week and chronic stud-ies with comparable dose levels� The observed differences were judged to be due to differences in dose selection and dose spacing or interexperimental variability or sensitivity of the study (low incidence or magnitude in studies with 4 dogs/sex/dose)�7 For one of the remaining eight substances (fosetyl Al), the limited details on clinical chemistry, haema-tology, or histopathology of the chronic study did not allow a meaningful comparison with the 13-week study� For two additional pesticides (cypermethrin, diflufenzopyr), there are pertinent rat studies with NOAELs/LOAELs compa-rable with the ones found in the 1-year dog study� In two other cases, the 13-week dog studies failed to detect effects that would be expected to occur prior to 13 weeks, namely cholinesterase inhibition (triazamate) and effects on thy-roid hormones (ethylenethiourea)� In addition, the 13-week triazamate study had no NOAEL (based on plasma choline esterase [ChE]), which would have led to an additional SF of 3 and thus making the result more protective than the NOEL of the 1-year study�

Only three compounds remained (etoxazole, hexazi-none, tebuconazole) for which selection of the NOAEL from the chronic dog study would possibly result in a lower RfD than if it were derived from the corresponding 13-week dog study� In the case of etoxazole, the difference was mainly in the LOAELs while the NOAELs were comparable, thus the RfD was not affected� The NOAELs for the subchronic and chronic dog studies conducted with hexazinone were 26 and 5 mg/kg, respectively� However, there is a chronic rat study with a NOAEL of 10�2 mg/kg body weight (bw), which in the regulatory perspective reduces ratio between subchronic and chronic NOAELs from 5 (dogs only) to 2 (all systemic tox-icity studies)� For tebuconazole, the difference in subchronic and chronic dog NOAELs is 7�7 versus 2�94 mg/kg bw, and

6Azafenidan, bifenthrin, bispyibac sodium, cadusaphos, clethodim, cyper-methrin, diflubenzopyr, ethylenthiourea, etoxazole, fenheximid, fosetyl Al, hexaconazole, hexazinone, prosulfuron, spinosad, tebuconazole, tebufenozide, thiophanate methyl, triazamate�7Azafenidan, bifenthrin, bispyibac sodium, cadusaphos, clethodim, fen-heximid,, hexaconazole, prosulfuron, spinosad, tebufenozide, thiophanate methyl�

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no other study is available with NOAELs or LOAELs lower than the chronic rat study� Thus in this case, the chronic RfD would have been higher by a factor of 2�6 if based on the 13-week dog study�

The authors concluded “Thus the present analysis indi-cates that a 13- week dog study would be adequate for identi-fication of a NOAEL or LOAEL that would be similar to that established from a chronic dog study except 3 pesticides (3/77 or 4%) of the cases evaluated.” And

“Thus, the results of this current retrospective analy-sis of studies on pesticides, when considered with the results of the analysis by Spielmann and Gerbracht (2001), show, with few exceptions, that a 13week dog study is as adequate as a 1-year dog study for identifica-tion of a NOAEL.”

A wider perspective is that in only 3 of 304 evaluations the results of the chronic dog studies may have influenced the derived reference doses� In the one case where only the LOAEL was different, it had no consequences, in the second the effect was mitigated from 5-fold to 2-fold by the data of a chronic rat study, and in the third case the difference was 2�6-fold, which is not impressive in view of the standard SF of at least 100, which is applied by default�

The position of the US EPA on the need for the 12-month dog study (Baetcke et al�, 2005) was discussed at the FIFRA Scientific Advisory Panel (SAP) Meeting of May 2005 (Anon, 2005)� The FIFRA SAP made the following recommendations “(1) increase the robustness of data analysis by including dog study datasets that were not used for the RfD determination; (2) conduct an analysis more representative of a prospective comparison through delineating the 13-week No Observed Adverse Effect Levels (NOAELs) and Lowest Observed Adverse Effect Levels (LOAELs) independent of the one-year study and establish data review criteria; (3) consider data analysis for separate classes of pesticides; (4) include additional back-ground information on RfD that provides better perspectives for reviewing the Agency position paper; and (5) revise the title of the Agency position paper to reflect the purpose of the data analysis.” The FIFRA SAP specified that “if the results of the analyses continue to indicate little added value from the one year studies, the Agency could move toward eliminating them on a stronger basis.”

In response to the FIFRA SAP comments, US EPA pro-vided additional analyses on pesticide chemicals in a document entitled “Length of Dog Toxicity Study(ies) that is Appropriate for Chronic RfD Determinations of Pesticide Chemicals” (dated March 20, 2006)� From a database of 330 compounds, 68 additional pesticides for which adequate studies over 13 weeks and ≥1 year were available were added to the original 42� Thus a total of 110 pesticides representing more than 50 classes were scrutinized� For half of the com-pounds (55), the chronic reference dose (cRfD) was based on dog studies; for the other half (55), the cRfD was based on rodent studies� The evaluation criteria were mainly to see whether data from 13-week studies (NOAELs, LOAELs)

would suffice to derive cRfDs in the absence of studies of longer duration� Again, the criterion to consider NOAELs or LOAELs as different was set at a factor of ≥1�5�

For 70 out of the 110 compounds, the 13-week and 1-year studies showed similar critical effects and the NOAELs and LOAELs were within a factor of 1�5 or less� For 40 pesticides, the NOAELs or LOAELs were lower than 1�5 times in the 1-year studies� A closer evaluation showed that for 10 pesticides, only the NOAELs differed by more than 1�5-fold, but not the LOAELs, and toxicities were simi-lar, thus the differences were attributed to dose spacing and selection� For another 10 pesticides, the subchronic studies would have been adequate to characterize toxicity and identify a protective NOAEL� For three pesticides with missing NOAELs in the subchronic studies, the 10 times lower extrapolated NOAEL was protective of the NOAELs found in the chronic studies� For eight more pesticides (ethylene thiourea, dinotefuran, epoxiconazole, fluazifop-buityl, fosetyl aluminium, mesosulfuron methyl, pyrimeth-anil, and spirodiclofen), there were various other plausible reasons to conclude that the chronic dog study would not have altered the outcome of the reference dose� New rel-evant toxicities found in the 1-year studies and not seen in the 13-week studies were rare (below 2%; cypermethrin, fosetyl aluminum)�

This left nine pesticides with differences in NOAELs or LOAELs that could not be ascribed to dose selection or spac-ing� For five out of these nine (bifenthrin, cypermethrin, bispyribac, hexazinon, tebuconazol), the cRfD was based on the results of the 1-year dog study� For the two pyrethroids, the rat data on neurotoxicity would have provided NOAELs and LOAELs adequately protective of any neurotoxicity seen in the chronic dog study, in particular if allometric scaling to account for the difference in overall body size had been applied� For bispyribac, in the absence of a chronic dog study the NOAEL and LOAEL from the chronic rat study, which were very similar to those of the 1-year dog study, would have been used with practically no impact on the cRfD� A similar situation was identified for hexazinon� For tebuconazole, the toxicity endpoints were similar between the two dog studies, and in the absence of a chronic dog study, the chronic rat study NOAEL would have been used and was considered to be protective�

For the other four (fluazinam, mancozeb, thiamethoxam, thiram), the cRfD was based on results of rodent (rat) studies� Therefore, the results of the chronic dog studies (or absence thereof) were not critical for the risk assessment as this was driven by the results of the rodent studies�

The study concludes, “This larger analysis supports the conclusion that longer-duration studies (one year) in the dog do not result in appreciably lower NOAELs or identify new effects for the majority of chemicals when compared to the shorter-duration study 13-week study in this species. (……) Even for the 8% where there are indications that a 1-year dog toxicity study would potentially provide a lower LOAEL than a 13-week study for purposes od RfD derivation, differences between LOAELs and NOAELs between the two dog studies

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were small (4 fold or less). It is unclear to what extent these small differences in LOAELs are meaningful from a practical standpoint relative to the 100-fold default uncertainty factor commonly used in calculating the RfD. In no case, did these small differences have a regulatory impact on pesticide risk assessments. As a result of the larger retrospective analysis dis-cussed above, OPP is moving to eliminate the routine require-ment of the 1-year dog study while retaining the 13-week dog study.”

In their “Response To Comments to Part 158 Rule Proposed March 11, 2005” (Docket ID: OPP-2004-0387), US EPA further explained, “Thus, reliance on the required chronic rodent studies, two-generation rat reproductive study, and the thirteen-week dog toxicity study is generally expected to provide an adequate basis for chronic RfD derivation in pes-ticide risk assessment. EPA acknowledges that there may be situations where a longer duration dog toxicity study may be warranted when a pesticide chemical is highly bioaccumulat-ing (e.g. builds up in body fat) and is eliminated so slowly that it does not achieve steady state or sufficient tissue concentra-tions to elicit an effect during a 90-day study. EPA anticipates that this situation will be infrequent since current pesticides are not usually designed to be highly persistent and bioac-cumulating. If such a chemical is encountered, EPA would require the appropriate tier 2 metabolism and pharmacoki-netic studies to more precisely evaluate bioavailability, half life, and steady state and determine if a longer duration dog toxicity study is needed.” As a consequence of this, US EPA subsequently removed the study from the list of studies required to support pesticide approvals (CFR 158�500, 26� Oct 2007, p 60976)�

The pharmaceutical industry has also discussed the reduction and refinement of dog studies in a similar con-text� Retrospective studies were published by Contrea et al� (1993) and DeGeorge et al� (1999), and numerous discussion panels and workshops have been held (Broadhead et al�, 2000; Smith et al�, 2002; Smith, 2006)� Recommendations have been aimed at harmonizing approaches and to reduce requirements such that only a single dog study of a longer duration of 9–12 months is performed, with interim data at 3 and 6 months based on biomarkers� This recommendation was based on the finding that in several case studies, data from longer studies were needed in order to identify addi-tional organ system toxicity not seen in 6-month studies or a shift in the dose-response for toxic effects�

As discussed by Baetcke et al� (2005), “Caution should be exercised, however, before comparing results of these analyses of toxicity tests performed in dogs with pharmaceuticals with results of dog studies conducted with pesticides. In the case of pharmaceuticals, it is important to characterize all pos-sible adverse effects that may occur in humans treated with pharmacological doses. Thus, the recommendations to extend dog studies on pharmaceuticals beyond 6-months appears to be based largely on additional effects that were observed at any dose in chronic dog studies but not in 6- or 9-month dog studies; there appear to be few examples in the literature reports where “new findings” affected the margins of safety.

In contrast, results of dog studies with pesticides are used pri-marily to identify dose levels below which no adverse health effects are likely to occur in humans exposed to environmen-tal levels of pesticides. Although additional effects observed above a LOAEL may support the application of additional uncertainty factors when RfDs are derived, the current retro-spective analysis did not identify a single case where only the result from a dog study would lead to the application of an additional uncertainty factor to a NOAEL.”

3. Synthesis of the data

The present paper focuses on four independent reviews, Spielmann and Gerbracht (2001), Baetcke et al� (2005), US EPA (2006), and Doe et al (2006), which considered the impact of the 1-year dog study relative to the 13-week study on establishment of reference doses for pesticide products� Each paper has its merits as well as some limitations with regard to the size of the databases and the methods used� However, each arrived at similar conclusions to limit test-ing of pesticides in dogs to studies of 3 months’ duration� A further synthesis of the combined data of the four papers provides the weight of evidence in support of the final conclusions�

3.1. Conceptual approaches/differencesThe reviews had somewhat different aims and also different databases� Therefore, it is not surprising that there are also some conceptual differences in the approaches� Spielmann and Gerbracht (2001) based their review(s) on a large and comprehensive number of original studies on file at the German BgVV, now BfR� The inclusion/exclusion criteria were not fully detailed� The coding of compounds was prob-lematic and did not allow a comparison with the databases used by the other authors, and it was not easy to correlate the numbers in the review with the extensive tables�

The review performed by Baetcke et al� (2005) was based on the large and comprehensive database of registered com-pounds by the US EPA and was then reduced in view of regu-latory relevance to 77 (42 + 35) compounds for which the dog data had influenced the derivation of the reference doses� Thus, compounds without actual registration (de-registered or not yet approved) are excluded� This reduces the data-base to some extent, but assures on the other hand that the criteria along which the evaluations were performed are in line with current standards� In the follow-up analysis by US EPA (2006), out of 330 regulated pesticides 110 were further examined because for these there were adequate 13-week and 1-year studies available� This review considered differ-ences in the results of the different study durations in dogs and examined the potential impact of the absence of chronic dog data�

The SABRE database, upon which the Doe et al� (2006) review is based, was considered to be a representative sam-ple of the pesticide database from the US EPA� The number of compounds examined (26–28) was relatively small� This was due to the fact that many more questions relating to

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systemic toxicity of pesticides were examined; comparisons between the 3-month and 1-year dog studies were only one part of the evaluation�

Therefore, two databases were actually considered by the above authors—the German SET/BfR (updated in 2004) and the US EPA, the latter being considered from various angles, on the basis of a representative sample, when studies in dogs were the basis for deriving reference doses and the potential impact of absence of chronic dog data� Therefore it can be

assumed that the great majority of the global database on relevant pesticide data with dogs has been considered and evaluated�

Further comparative aspects of concepts and databases of the publications are listed in Table 3�

3.2. Size of the entire datasetAs shown in table 4, the largest list of named compounds is contained in the Gerbracht and Spielmann (1998)

Table 3. Comparative considerations of reviewed publications with regard to study duration.

Database Inclusions/exclusions Comments Key conclusion

Gerbracht and Spielmann (1998)

232 pesticides on file in BgVV (BfR), 216 with suitable data-bases Registration status not specified Original reports

16 compounds eliminated due to quality/ comparability concerns

Coded results only Little explanation how studies were assessed and summed up Study listed, as it is essential for the two below

Dog is needed and valuable as second species.

Spielmann and Gerbracht (2001)

172 of the above, dog data from subacute, subchronic, and chronic studies. Registration status not specified Original reports

Availability of data sets in dogs 4- or 13-week studies versus 52/104-week studies

Unclear how studies were assessed and summed up, compares in certain cases with data from rats, but not on the consequence on RfDs

Safety testing of pesticides in dogs should be limited to subchronic (13-week) studies.

Box and Spielmann (2005)

Primarily BfR data (SET/BfR study) plus additional extensive literature search

As within the previous publications

Alternative proposals for a trigger for 12-month dog study or additional UF instead of 12 month dog study

Routine testing should be limited to 3-month studies in dogs.

Doe et al. (2006) SABRE Database. Choice of com-pounds randomly selected to be representative of different pesti-cide classes on file in US EPA. Specifically built for ILSI ACSA project, original data accessible. Total 65 chemicals 26–28 thereof had complete data sets and detailed evaluations

Completeness of data set, regulatory assess-ments available. Thus only compounds with registrations (actual or past)

Most complete consideration, but on (representative) small data base only. Supports new testing paradigm for pesticides.

A 12-month dog study is not needed. There is little difference in dos-sier without 12-month dog study.

Baetcke et al. (2005) Originally 304 compounds in US EPA, taken those on which RfDs were based on dogs. Original data accessible. Had 4/13-week and 52/104-week data

Only registered (= fully assessed) compounds, no deregistered, no “ un-registration” compounds

Review performed from regula-tory perspective with regard to 3/12 months. No consideration of dossier without 12-month study.

A 3-month dog study and chronic rodent studies are an adequate basis to character-ize NOAEL and LOAEL for pesticides.

US EPA (2006) Originally 330 compounds in US EPA, chosen those with adequate studies in dogs with a duration of ≥1 year and 13 weeks. Original data accessible.

Only registered (= fully assessed) compounds, no deregistered, no “ un-registration” compounds

Review performed from regula-tory perspective and consid-ered consequences of absence of chronic dog studies

Extension of a dog study beyond a 13-week duration does not provide additional, essential information. Reliance on chronic rodent and 13-week dog studies sufficient for cRfD derivation in pesticide risk assessment.

Table 4. Numerical summary of the key reviews.

SET/BfR* Doe et al. (2006) Baetcke et al. (2005) EPA (2006) Total

Total compound database 232 28 (out of 65) 304 330

Compounds with studies in dogs or dog crucial to RfD setting

172 26 77 110 n/a due to overlap

Compounds not in Spielmann & Gerbracht studies 13 45 63

No. of compounds not mentioned in other reviews 172* 13 45 (45**) 230

No. of compounds for which 12-month study is con-sidered relevant

3/26 tebuconazole, lactofen, butylate

3/77 tebuconazole, etoxazole hexazinone

0 7 + 3 + 3 − 1 = 12

*Gerbracht and Spielmann (1998), Spielmann and Gerbracht (2001), list taken as point of departure.**Other than Baetcke (2005).

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publication, although specific experimental results were coded� The other three key publications, Baetcke et al� (2005), US EPA (2006), and Doe et al� (2006), name only the compounds examined closer� It is logical, therefore, to take the list of compounds from the SET/BfR study (Gerbracht & Spielmann, 1998) as basis and assess the other publications for any additional compounds�

The simple summing of the total numbers of compounds in the database is certainly not correct, as there are likely overlaps� The available data in the publications allow at least to identify which compounds have been scrutinized in which review and to estimate the degree of overlaps�

The SET/BfR study was based originally on 232 com-pounds, the Baetcke et al� (2005) study with the US EPA database with 304 compounds� Simply adding these would be incorrect due to the likely high degree of overlap

anticipated� However, a range can be estimated� The addi-tion of the 172 compounds examined by Spielmann and Gerbracht (2001) and the 13 and 45 by the other reviews that are not mentioned in the SET/BFR database result in a total of 230 substances, which is the minimum number reviewed among all three publications� Of the 77 com-pounds listed and examined by Baetcke et al� (2005) and for which the dog studies were crucial for RfD setting, 45 were not mentioned in the SET/BfR database� This is more than half of the 77 and points to a partially different data-base� Half of the US EPA database of 304 would be about 150� This number might be added to the 232 from the SET/BfR database, which points to an estimated range maxi-mum of about 380� A similar result would emerge from the compounds used in the US EPA (2006) review� Sixty-three of the listed 110 compounds are not mentioned in the SET/

Table 5. Compounds with findings potentially supporting 1-year study: Assessments by Doe et al. (2006) and Baetcke (2005).

Compound

NOAEL 1-y dog mg/kg bw

NOAEL 3-mo dog mg/kg bw

NOAEL Other species mg/kg bw

LOAEL 12-mo dog mg/kg bw

LOAEL 3-mo dog mg/kg bw

Magnitude of not having 1-y dog study

Hexaconazole 2 5 3 (3-month rat) 10 <2-fold

2 5 10

Acetochlor 2 10 10 (2-y rat) 12/40 2-fold with 1 study

2 2 (int) 10

Butylate 5 (25)* 45 32 (3-mo rat) 25 (100)* 9 (<2)-fold*Tebuconazole 2.9 73 5 (2-y rat) 4.4 <2-fold

2.94 7.5 7.4 (2-y rat) 4.39 2.5-fold

Lactofen 0.79 75 0.79 (2-y rat) 4 <2 fold

Etoxazole 4.62 4.62 (int diet) 23.5 23.5 <2-fold

5.33 (3-m caps study)

53.7

Hexazinone 5 (diet) 25.9 (diet) 10.2 (2-y rat) 37.6122.5 (37.6) 2-fold

Fosethyl Al** 250 274 400 (2-y rat) 500 <2-fold

250 274 500 1309

Iprodion** 17.5 60 6.1 (2-y rat) <2-fold

Rimsulfuron** 81.8 9.63 342.4193 3-mo 193 <2-fold

Comments:

Hexaconazole Difference between NOAELs of 3- and 12-month studies, 2.5-fold. However, NOAEL of 3-month rat = 3 mg/kg, thus RfD would be change by factor of 1.25 only. Identical levels in Doe and Baetcke.

Acetochlor Doe reports two 1-year dog studies, one within 2-fold. Baetcke reports NOAEL of 3-month interim the same as the final 12-month NOAEL. Thus some difference in interpretation, though with similar result.

Butylate: Doe assesses data as “difficult to understand”, because 1-year dog (5 mg/kg) is clearly lower than the other NOAELs (3-month dog: 45 mg/kg; 3-month rat: 32 mg/kg; 2-year rat: 100 mg/kg). However, a reinspection of the data indicated that the NOAEL of the 1-year dog study should be 25 mg/kg bw, thus reducing the magnitude to 1.8-fold.

Tebuconazole Doe reports 3-month dog NOAEL higher than other NOAELs, not dose selection artefact. The next relevant NOAEL is from rat 2-year at 5 mg/kg, thus still within 2-fold.

Baetcke reports different NOAEL for 3-month dog = 7.5 mg/kg, and a slightly different 2-year rat NOAEL of 7.5 mg/kg, thus RfD 2.5-fold higher.

Compound listed by Spielmann and Gerbracht (2001) but not specifically mentioned.

Lactofen Doe assesses results not as a selection artefact. However, NOAEL 2-year rat = 0.79 mg/kg, thus RfD would not change.

Etoxazole Interim result and separate 3-month study have comparable NOAEL (5.33 or 4.62 mg/kg) as 1 year study, thus no change in RfD.

Hexazinone Difference in NOAEL of 5.2-fold between 1-year and 3-month study, but mitigated by 2-year rat study with NOAEL of 10.2 mg/kg, thus a difference in RfD of 2.04-fold.

Fosethyl Al Difference smaller than factor 2, according to Baetcke, insufficient detail. With NOAEL of 2-year rat, difference smaller than 2-fold.

Iprodion Difference between 3-month and 1-year dog NOAELs 3.4-fold, but ADI driven by 2-year rat study with lower NOAEL, thus below 2-fold.

Rimsulfuron 1-Year dog study has higher NOAEL than 3-month dog study.

*Reinspection of the data indicated that the NOAEL of the 1-y dog study should be 25 mg/kg bw, thus reducing the magnitude to 1.8-fold.**Compounds not raised by Doe et al. (2006) or Baetcke et al. (2005), but mentioned in Spielmann and Gerbracht (2001).

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10 W. Kobel et al.

BfR database, which is practically the same ratio, thus not changing the estimated range�

Of the 77 named compounds in the Baetcke et al� (2005) review, 45 are not listed in any of the two other papers� Of the remaining 32 compounds, only 26 are on the list of Gerbracht and Spielmann (1998) and 9 appear in Doe et al� (2006), 3 appear in both reviews� This is further evidence that the SET/BfR database is different from the US EPA database used by Baetcke et al� (2005) and that the two overlap only in parts� A similar picture emerge when counting compounds from the US EPA (2006) review� Sixty-five of 110 are listed only in this review (or Baetcke, 2005)�

As the SABRE database built for the Doe et al� (2006) investigation is also US EPA based, one could expect that all compounds for which the chronic dog study is relevant would also appear in Baetcke et al� (2005)� Of the five com-pounds identified by Doe et al� (2006), only acetochlor, hexaconazole, and tebuconazole are listed in Baetcke et al� (2005), whereas lactofen and butylate (discussed later) are not on the list� The US EPA (2006) review list hexaconazole and tebuconazole only� This is surprising, given the relative similarity of databases and is not explained in any of the reviews�

Adding the studies in which the 12-month dog study may have shown a relevant result not seen in the remain-ing database, Spielmann and Gerbracht (2001) contribute 7 coded compounds� In the two other reviews with US-based data there are 3 + 3 compounds with one overlap, thus in total 5� Thus a very conservative approach is to add the 7 and the 5, which results in a total of 12 for both databases� The US EPA (2006) review does not add further compounds to this number, This total should be weighed against the total number of at least 300 compounds scrutinized�

3.3. How were compounds judged in the reviews?Pesticides of particular interest are those for which the chronic dog study may be of relevance for the derivation of reference doses� Whereas the Spielmann and Gerbracht

(2001) review only allows limited identification of indi-vidual compounds, the other two papers named those substances potentially supporting chronic dog studies� The findings as extracted from the reviews are listed in Table 5 and 6�

There are similarities among the discussed findings of compounds with potentially relevant findings between Doe et al� (2006) and Baetcke et al� (2005)�

For the seven compounds identified/discussed by either Doe et al� (2006) or Baetcke et al� (2005), the relevance of the 1-year dog data were assessed with a view of the deri-vation of a chronic reference dose (RfD), the equivalent of the ADI� Without the 1-year dog data, for six compounds the RfD would change less than 2-fold, for another two, the change would be 2- and 2�5-fold� Only one compound, butylate, would have a RfD which is 9 times higher (45 ver-sus 5 mg/kg bw) and was judged as “difficult to understand”� However, inspection of the data (IRIS; http://www�epa�gov/ncea/iris/subst/0215�htm) within this work revealed the likely explanation� The 13-week and 1-year studies for butylate were conducted 20 years apart� The 13-week study was performed prior to the establishment of test guidelines or Good Laboratory Practices (GLPs) (1967) and was via the diet compared to via capsules for the 1-year study� In addi-tion, the NOAEL for the 1-year study is probably 25 mg/kg bw/day, as unequivocal treatment-related effects occurred only at 100 mg/kg bw/day; 25 mg/kg bw/day was deemed an effect level based solely on relative liver weight in males (21% above control), but with no associated clinical chemistry or histopathological changes and no difference in females (which were as sensitive to liver effects as males at the high dose level)� This is probably not a treatment-related effect, and is not considered as adverse per se� Accordingly, the fac-tor between the two NOAELs would be reduced to 1�8 and thus all of the differences between the NOAELs in 13-week and 1-year studies are within a factor of ~2�

Fosetyl Al, iprodion, and rimsulfuron are also listed in Table 6a because they are named in Spielmann and

Table 6. Compounds with findings potentially supporting 1-year study: Comments by Spielmann and Gerbracht (2001).

Compound Reason for mentioning New findings in 1-year study Comment/conclusionAssessments by Doe et al. (2006) and Baetcke et al. (2005)

Dichlofluanid In 8/54 fungicides Testicular and adrenal degeneration —

Among the 7/141 No gravimetric measurements/histopath in 3-month study. Urinary alteration also in rats.

Fosethyl Al* In 8/54 fungicides Testicular degeneration Insufficient detail, ADI within 2-fold

Pyrazophos In 8/54 fungicides Specific gravity of urine decreased, calcification of Bowman capsule, effects minimal, also seen in rats

—

Among the 7/141

Iprodion* In 8/54 fungicides Small granuloma and leucocyte infiltration of urinary bladder

Difference in dog studies 3.4 fold, but ADI within 2 fold

Bromuconazole, In 8/54 fungicides (adrenal & kidney) not important —

Diniconazole In 8/54 fungicides (reproductive system) not important —

Fluazinam In 8/54 fungicides (haematopoetic system) not important —

Trinexapac ethyl (7/141??) Hippocampus and lateral mid brain vacuolization. Biological significance unknown, not associated with pathological changes or overt neurological signs

—

Rimsulfuron* Among the 7/141 Mucosal hyperplasia of trachea minimal Reviewed by Baetcke, not commented.

*See also assessment in Table 5.

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http://www.epa.gov/ncea/iris/subst/0215.htm


Gerbracht (2001), yet they are also within the difference of 2 fold, if at all�

In the Spielmann and Gerbracht (2001) review, there were nine compounds identified� Three are certainly part of the “7 of 141” considered to potentially support the need for 12-month study� Seven belong to a subgroup “8/54 fun-gicides”� In these identified substances with findings, there appears to be some doubts as to the relevance of the differ-ences found� The comments indicate that the assessments made were rather conservative, meaning that the differences found and accepted were not always based on the same level of data� The three compounds certainly belonging to the “7/141”; the authors express some reservations, and for two more compounds also examined by the other authors, the RfD difference would stay within 2-fold� Thus it seems justified to assume that the seven compounds found by Spielmann and Gerbracht would be further reduced when assessed for the outcome of the RfD�

An even clearer picture was found by US EPA (2006)� Of the 110 compounds with adequate dog data sets, there were only 9 in which the NOAEL and/or LOAEL from the chronic dog study was lower than the one from the 13-week study� In the five cases (bifenthrin, cypermethrin, bispyribac, hex-azinon, tebuconazol), where the cRfD was actually based on chronic dog data, there were rodent studies with NOAELs/LOAELs comparable to those of the chronic dog study� For the remaining four compounds (fluazinam, mancozeb, thiamethoxam, thiram), the cRfD was based on rodent data, thus the chronic dog data were not critical and did not drive the risk assessment�

4. Conclusions/recommendations

It is well understood that for each of the reviews, there are limitations and doubts as to the comprehensiveness, as, for example, expressed in the SAP review of the Baetcke et al� (2005), which asked for a broader review including the entire database and not only those in which the dog drives the RfD� US EPA has considered this point and came in their expanded and updated review of 2006 to a much clearer conclusion that for pesticide risk assessment, dog studies should not be longer than 3 months� There is no doubt that a repeat-dose study of 3 months’ duration in dogs is needed and this point is not in dispute� However, the weight of evidence based on all of the reviews together, and in particular Spielmann and Gerbracht (2001), Doe et al� (2006), Baetcke et al� (2005), and US EPA (2006), allows the clear conclusion that a 12-month dog study in addition to a 3-month study is of little value and the requirement for this study should be eliminated from the list of mandatory studies to be performed in the safety assessment for pesticides� In the entire databases analyzed in the four main publications critically reviewed here, there are at most 3–4% of compounds in which the results of a 12-month dog study may have influenced a reference dose� The combined weight of evidence from the four reviews is considered clearly sufficient to drop the 12-month dog study for pesticides and limit the dog studies to 3 months� The fact

that the approaches for each review were different but came to very similar results strengthens the argument further�

In those cases in which the dog appears to be substan-tially more sensitive than the rat in 3-month studies or shows a different spectrum of findings, a thorough comparative evaluation for kinetics, allometric comparisons, and differ-ences in dynamics is indicated� Simply applying a generic or chemical-specific uncertainty factor is not an acceptable default�

The weight of evidence from the reviews individually, and even more so from their combined considerations, supports the conclusion that without compromising human safety routine, dog testing is needed but can be limited to studies of 3 months’ duration, for which improvements of the design should be considered, as suggested in some of the reviews� Therefore, based on a balanced combination of science and animal welfare considerations, those national requirements still insisting on a 12-month dog study for the registration of a pesticide should be updated as soon as possible to harmo-nize with the US EPA and the EU and eliminate the require-ments for dog studies beyond 3 months�

Appendix

List of the all pesticides of which toxicological profiles were evaluated


Doe et al. (2006)

Baetcke et al. (2005) US EPA (2006)

2,4 D

2,4,DB

Abamectine (I)

Acetamiprid

Acetochlor Acetochlor

Aldicarb (I/N/A)

Aldimorph (F)

Alpha-cypermethrin (I)

Amidosulfuron (H)

Amitraz (I/A) Amitraz Amitraz

Amitrole (H)

Anilazin (F)

Atrazine

Azafenidan

Azaconazole (F)

Azibenzolar-S-methyl

Azinophos-methyl (I/A)

Azinphos methyl

Azocyclotin (A)

Azoxystrobin (F) Azoxystrobin

Benalaxyl (F)

Bendiocarb (I)

Benfluralin

Benfuracarb (I)

Benomyl (F) Benomyl

Bensulide Bensulide

Bentazone (H) Benatazon Benatazon

Betacyfluthrin (I)

Table continued on next page.

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Bifenazate Bifenazate

Bifenox (H)

Bifenthrin (A/I) Bifenthrin Bifenthrin Bifenthrin

Bispyibac sodium

Bispyibac sodium

Bitertanol (F)

Boscalid

Bromadiolon (R)

Bromophos (I)

Bromoxynil (H) Bromoxinyl phenol

Bromoxinyl phenol

Bromopropylate (A)

Bromuconazole (F) Bromuconazole

Buprofezin (I)

Butocarboxim (I)

Butafenazil

Butylate

Cadusafos Cadusafos

Carbaryl Carbaryl

Carbendazim (F)

Carbetamide (H)

Carbofuran (I) Carbofuran

Carbosulfan (I)

Carboxin (F) Carboxin (no 1Y!)

Carfentrozonethyl

Chinomethionat (F,A)

Chlorethoxyfos Chlorethoxyfos

Chlorfenapyr (A,I) Chlorfenapyr Chlorfenapyr Chlorfenapyr

Chlorfenvinphos (I)

Chloridazon (H)

Chlormequat (W)

Chlorpropham (H) Chlorpropham

Chlorpyrifos (I)

Chlorpyrifos-methyl (I)

Chlorothalonil (F)

Chlorotoluron (H)

Clethodim Clethodim

Clodinafop (H) Clodinafop

Clofentezine

Clomazone (H)

Clopyralid (H)

Cloquintocet (S) Cloquintocet.mexyl

Coumaphos

Cyanamide (H)

Cyazofamid

Cycioxidim (H)

Cyfluthrin (I) Cyfluthrin Cyfluthrin

Cyhalofop-butyl

Cyhalothrin

Cymoxanil (F) Cymoxanil

Cypermethrin (I) Cypermethrin Cypermethrin

Cyproconazole (F)

Cypodinil (F) Cyprodinil (no 1Y!)

Cyprodinil


Doe et al. (2006)


Cyromazine (I)a

Dazomet (N)

Deiquat (H)

Deltamethrin (I) Deltamethrin

Demeton-S-mcthyl (I)

Desmedipham (H) Desmedipham

Desmetryn (H)

Diazinon (I) Diazinon

Dicamba

Dichlobenil (H)

Dichlofluanid (F)

Diclorprop-P (H)

Dicloran

Dichlorprop (H)a

Dichlorvos (1) Dichlorvos

Dicofol Dicofol Dicofol

Diclobutrazol (H)a

Diclofop-methyl (H)

Dicloran

Didecyldimethyl-ammonium (D?)

Diethofencarb (F)

Difenoconazole (F)

Difethialone (R)

Diflubenzopyr Diflubenzopyr

Diflubenzuron (I) Diflubenzuron Dilubenzuron

Diflufenican (H)

Dikegulac (W)

Dimefuron (H)

Dimethenamid (H)

Dimethoate (I) Dimethoate

Dimethomorph (F) Dimethomorph

Diniconazole (F?)

Dinotefuran

Diquat dibromide

Disulfoton

Dithianon (F)

Diuron (H)

Endosulfan (I)

Emamectin

Epoxiconazole (F) Epoxiconazole

EPTC (H)

Ethephon (W)

Ethiofencarb (I)

Ethion

Ethirimol (F)

Ethofumesate (H)

Ethoprop

Ethoprophos (N,I)

Ethylenthiourea a Ethylenethiourea ETU

Etoxazole Etoxazole

Famoxadone

Fenamiphos

Fenarimol (F)

Fenazaquin (A)

Table Continued.


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Fenbuconazol (F)

Fenbutatin-oxide (A)

Fenchlorazole (S)

Fenfuram (F)

Fenhexamid Fenhexamid

Fenitrothion

Fenoxaprop-ethyl (H)

Fenoxaprop-P (H)

Fenoxycarb (I)a

Fenpiclonil (F)

Fenpropathrin (I) Fenpropathrin Fenpropathrin

Fenpropimorph (F)

Fenpyroximat (A) Fenpyroximate

Fentin-acetate (F)

Fentin-hydroxyde (F)

Fenthion (I)

Fenvalerate (A/I)

Fipronil

Flonicamid

Fluazifop-p-butyl (H)

Fluazifop-butyl

Fluazinam (F) Fluazinam

Flucarbazone Flucarbazon

Fludioxonil (F) Fludioxonil Fludioxonil

Flufenpyr

Flumioxazin

Fluoroglycofen (H)

Fluoxastrobin Fluoxastrobin

Flurochloridone (H)a

Fluroxypyr (H)a

Flurprimidol (W)

Flusilazole (F)

Flutriafol (F)

Fluvalinate (I)a

Fosetyl (F) Fosetyl AL Fosetyl Al Fosetyl Al

Foramsulfuron

Fuberidazole (F)

Furathiocarb (I)

Glufosinate (H) Glufosinate ammonium

Glufosinate ammonium

Glyphosphate (H)

Glyphosphate-trimes (H)

Guazatine (F)

Halosulfuron methyl

Hexaconazole Hexaconazole Hexaconazole

Hexazinone Hexazinone

Hexithiazox (A)

Hymexanol (F)

Imazalil (F) Imazalil

Imazetapyr

Imidacloprid (I)

Indoxacarb


Doe et al. (2006)


Iodosulfuron Iodosulfuron

loxynil (H)a

Iprovalicarb

Iprodione (F) Iprodione

Isofenphos (I)

Isoproturon (H)

Isoxaben (H)

Isoxadifen Isoxadifen

Kresoxim-methyl (F)

l-Cyhalothrin (I)a

Lactofen

Lenacil (H)

Lindane (I)

Linuron (H) Linuron

Mancozeb (F) Mancozeb

Maneb (F)

MCPA (H) MCPA

Mecoprop-P (H)

Mefenpyr-diethyl

Mepanipyrim

Mepiquat chloride

Mepiquat

Mesosulfuron Mesosulfuron

Metalaxyl (F)

Metaldehyde (M)

Metam-sodium (N)

Metamitrone (H)

Metazachlor (H)

Methabenzthiazuron (H)

Methamidophos (1)

Methidation (A/I) Methidathion

Methiocarb (1)

Methoprene (1)

Methoxyfenozide

Methyl-isothiocyanat (N)

Methyl- parathion

Metiram (F)

Metobromuron (H)

Metolachlor (H) Metolachlor Metolachlor Metolachlor

Metosulam (H)

Metribuzin (H)

Metsulfuron-methyl (H)

Mevinphos

Myclobutanil (F) Myclobutanil

Napropamide (H)

Nitrapyrin

Nitrothale-isopropyl (F)

Nuarimol (F)

Omethoate (1)

Oxydemeton-methyl (A/I)

Oxydemeton methyl

Table Continued.


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Paraquat (H) Paraquat Paraquat

Parathion

Parathion-ethyl (I)

Penconazole (F)

Pencycuron (F)

Pendimethalin (H)

Penoxsulam

Pentachlorphenol

Permethrin (I)

Phenmedipham (H)

Phorate Phorate

Phosphamidon (I/A)

Phostebupirim Phostebupirim

Phoxim (I)

Pinoxaden

Piperonyl-butoxide (S)

Prallethrin

Primicarb (I)

Prallethrin Prallethrin

Primiphos-methyl (I/A)

Primisulfuron (H) Primisulfuron methyl

Primisulfuron methyl

Prochloraz (F)

Procymidone (F)

Propachlor Propachlor

Prohexadion Prohexadion

Propamocarb (F)

Propaquizafop (H)

Propiconazole (F) Propiconazole

Propineb (F)

Propoxur (I)

Propyzamide (H)

Prosulfocarb (H)

Prosulfuron Prosulfuron

Pymetrozine

Pyraflufen-ethyl

Pyrazophos (F)

Pyrethrum (I)a

Pyridate (H) Pyridate

Pyridiben

Pyrifenox (F)

Pyrimethanil (F) Pyrimethanil

Pyriproxyfen

Quinoclamine (H)

Quizalofop-ethyl (H)

Rimsulfuron (H) Rimsulfuron Rimsulfuron

Simazine

Spinosad Spinosad

Spirodiclofen

Spiroxamine

Sulcotrione (H)

Sulfentrazone

Sulfosulfuron

Sulfotep (A/I)


Doe et al. (2006)


Sulfosate Sulfosate

Sulprofos

Tau-Fluvinatea

Tebuconazole (F) Tebuconazole Tebuconazole Tebuconazole

TCP

Tebufenpyrad (A)

Tebufenozide Tebufenozide

Teflubenzuron (I) Teflubenzuron

Tefluthrin (I)

Tepraloxydim

Terbufos (I) Terbufos

Terbuthylazine (H)

Terbutryne (H)

Thiabendazole (F) Thiabendazole

Thiacloprid

Thiafansulfuron (H)a

Thiamethoxam

Thiapenthenol (W)

Thiodicarb (M/I)

Thiophanate-methyl (F)

Thiophanate methyl

Thiophanate methyl

Thiram (F) Thiram

Tolcofos-methyl (F)a

Tolyfluanid (F)

Tralkoxidim Tralkoxidim

Triadimefon (F) Triadimefon Triadimefon

Triadimenol (F) Triadimenol Triadimenol

Triallate (H) Triallate

Triapenthenol (W)

Triasulfuron (H)

Triazamate Triazamate

Triazophos (I)

Triazoxide (F)

Tribenuron (H)

Tribufos

Trifloxysulfuron-sodium

Triflumuron (I)

Tridemorph (F)

Trifloxystrobin

Trifluralin (H)

Triflusulfuron (H)

Trisulfuron-methyl

Triforine (F)

Trinexapac-ethyl (H?)

Vinclozolin (F) Vinclozolin

Zetacypermethrin (I)

Zineb (F)

Zoxamide Zoxamide

Note. Abbreviations from “Spielmann” column: A, Acaricide; F, fungicide; H, herbicide; 1, insecticide; M, molIuscicide; N, nematicide; R, rodenticide; S, synergist for insecticides; W, growth regulator.aTest compound eliminated from the study due to the quality and/or lack of comparability.Italic in Baetcke column are NOAELs and LOAELs from 1- or 2-year dog studies with interim data (1–3 months).

Table Continued.

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Acknowledgements

The authors appreciate the valuable support of Mrs� Ingeborg Gerstner of BASF for providing the literature needed for this review�

Declaration of interest

The work of the primary author (WK) of this paper was supported by the European Crop Protection Association (ECPA)� WK is the principal in the firm, ToxAdvice GmbH� The firm provides advice on toxicology issues to private firms, including firms developing, registering, manu-facturing, and marketing pesticides� Eight of the other authors, IF, RB, RL, KB, WB, PB, BS, and BvR, are employed as shown on the cover page by firms that develop, regis-ter, manufacture, and market pesticides� These firms are members of the ECPA� The other author, HS, is employed by a University and is also affiliated with a Germany Federal Institute, which is concerned with risk assessment matters� He did not receive any honorarium or reimburse-ments� The authors worked as a Committee of the ECPA in preparing the analyses reported in the paper� The authors alone are responsible for the content and writing of the paper�

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Anonymous (2006). CFR 158.500, 26. Oct 2007, p 60976

Baetcke KP, Phang W, Dellarco V (2005). A Comparison of the Results of Studies on Pesticides from 12- or 24-Month Dog Studies with Shorter Duration. US EPA, 4/7/05. Available at http://www.epa.gov/scipoly/sap/meetings/2005/may2/dogstudymay05.pdf.

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