I Executive summary of BPA Consortium comments to the CLH Proposal 3

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1 BPA Cnsrtium cmments n the Prpsal fr harmnised classificatin and labeling (CLH Reprt) n Bisphenl A (BPA) prepared by ANSES Versin 2.0, dated 17/07/2013 Cntents I Executive summary f BPA Cnsrtium cmments t the CLH Prpsal 3 II BPA Cnsrtium s cmments n the CLH prpsal 5 A Multiple multigeneratin studies cnfirm that BPA is nt a reprductive txicant 5 B The CLH prpsal fails t engage in a weight f the evidence analysis as required by ECHA s Guidance 6 1 The CLH prpsal des nt cnsider all available infrmatin 7 C D 2 The CLH prpsal des nt fllw the CLP Regulatin Guidance requirement that Bth psitive and negative results shall be assembled tgether in a single weight f evidence determinatin. 3 The CLH prpsal fails t fllw the CLP Regulatin requirement that the cnsistency f the data shall be given apprpriate weight 4 The CLH prpsal fails t fllw the CLP Regulatin requiring that The quality f the data shall be given apprpriate weight 5 The infrmatin n reprductive endpints reprted in the CLH prpsal is incnsistent thrughut the prpsal 6 Differences in strain sensitivity are mentined thrughut the CLH prpsal, but the statements are cntradictry, and relevant infrmatin n strain sensitivity is nt included 7 The CLH prpsal inapprpriately rejects the results f guideline studies based n validated prtcls, yet accepts withut questin the results f explratry studies 8 Epidemilgical studies referenced in the CLH prpsal prvide n evidence t supprt stricter classificatin f BPA Statements abut the multigeneratin animal studies upn which regulatrs have relied (Tyl et al 2002 and 2008a) are incnsistent, incrrect and cntrary t evaluatins f these studies Recent and imprtant infrmatin available n a new study cnducted at the US FDA Natinal Center fr Txiclgical Research (NCTR) is nt included in the CLH prpsal Page 1 f 23

2 E The CLH prpsal is nt cmprehensive in cnsidering the available txickinetics data 17 F Reference t baby bttles as surce f expsure is utdated 18 G The CLH prpsal refers t an incrrect industry self-classificatin in chapter 1.7 Current self-classificatin and labelling 19 III Overall Cnclusin 20 IV Annexes 21 A B C D E CLH prpsal cntains incnsistent and incrrect infrmatin n male and female endpints CLH prpsal cntains incnsistent and incrrect infrmatin n Tyl et al (2002 and 2008a) NCTR Evaluatin f the txicity f Bisphenl A (BPA) in Male and Female Sprague- Dawley Rats Expsed Orally frm Gestatin Day 6 thrugh Pstnatal Day 90 Overview f relevant studies fr classificatin f Bisphenl A (BPA) Review f the epidemilgy studies described in the ANSES 2013 reprt n harmnized classificatin and labeling f Bisphenl A V References 22 Page 2 f 23

3 I. Executive summary f BPA Cnsrtium cmments t the CLH Prpsal These cmments and attachments are the cmments f the Bisphenl A REACH Cnsrtium (BPA Cnsrtium), which represents mre than 30 f the main prducers, imprters and users f BPA in Eurpe. After careful review f the prpsal in the CLH dssier, we have a number f cncerns. The case has nt been made that BPA merits classificatin as Categry 1B (presumed reprductive txicant) under the CLP Regulatin. In fact, a review f the relevant studies shws that effects n animal fertility nly ccur at high dses f BPA and that, rather than being selective reprductive effects, they are merely related t systemic txicity. The CLH prpsal is nt cnsistent with the prcedure utlined in ECHA s Guidance n the preparatin f dssiers fr harmnised classificatin and labeling (ECHA 2010) 1 which directs the use f a weight-f-evidence apprach fr cmpunds with a large database, such as BPA. The CLH prpsal des nt cnsider all available infrmatin; des nt fllw the CLP Regulatin standard regarding the request that Bth psitive and negative results shall be assembled tgether in a single weight f evidence determinatin; and fails t fllw the CLP Regulatin in that The quality f the data shall be given apprpriate weight. The CLH prpsal selectively relies nly n studies, assessments, and the 1 ut f self-classificatins that supprts its prpsal and, therefre, prtrays an inaccurate and incmplete picture f the state f the science n BPA. Infrmatin is nt cmprehensive and incnsistent thrughut the reprt. Statements related t the value f regulatry guideline studies cmpared t the value f explratry studies are biased. Statements n multigeneratin animal studies upn which regulatrs have relied (Tyl et al 2002 and 2008a) are incnsistent, incrrect and incmprehensible. Reference f ne industry self-classificatin ut f is clear evidence f cherry picking infrmatin and ignring cntrary infrmatin. Recent (pst December 31, 2012) and imprtant scientific research frm gvernment agencies was nt cnsidered. These gvernment studies d nt supprt a classificatin f BPA as a Categry 1B Reprductive Txicant. Given the abve, the dssier shuld be rejected as nt supprting a classificatin f BPA as Categry 1B reprductive txic. The CLH prpsal des nt fulfil the criteria utlined in ECHA s Guidance n the preparatin f dssiers fr harmnised classificatin and labeling 1 EU Regulatin (EC) N 1272/2008 n Classificatin, Labelling and Packaging f Substances and Mixtures, the CLP- Regulatin, entered int frce n 20th January 2009 Page 3 f 23

4 (ECHA 2010) because it fails t cnsider the quality f the data and it fails t cnsider all f the data in a weight f evidence analysis. As can be seen frm the BPA Cnsrtium cmments and frm assessments f BPA cnducted by ther gvernment regulatrs, when all high quality scientific studies n BPA have been cnsidered and a weight f the scientific evidence evaluatin is cnducted, it will clearly demnstrate that BPA is nt a selective reprductive txicant. In cnclusin, there is n basis t change the classificatin f BPA t Categry 1B. Page 4 f 23

5 II. BPA Cnsrtium s cmments n the CLH prpsal A. Multiple multigeneratin studies cnfirm that BPA is nt a reprductive txicant Many multigeneratin studies n BPA prvide ample data t demnstrate that classificatin f BPA as a Categry 1B reprductive txicant is nt supprted. Three prir assessments f BPA by Eurpean regulatrs assessed the then available data and determined that BPA was nt a selective reprductive txicant. Mre recent studies, including an extensive study by US FDA Natinal Center fr Txiclgical Research (NCTR), simply add t the verwhelming scientific evidence that BPA is nt a selective reprductive txic and that BPA des nt meet the criteria fr classificatin as a Categry 1B reprductive txicant. The histry f Eurpean regulatry assessments f BPA is instructive. In 2002, after a lng and thrugh weight f the evidence analysis f the then available data n the ptential effects f BPA n animal fertility and develpment, BPA was classified by the Member States as reprductive txicity Categry 3 fr fertility (Directive 67/548/EEC; R 62.) The cnclusin reflects the Member State s assessment that the reprductive effects frm BPA (e.g. reductin f litter size) were nly bserved when significant txic effects t the whle bdy were bserved (e.g. influence n bdy weight and effects n liver and kidneys.) Thus, the Member States wrking grup cncluded that the effects n fertility shuld be regarded as related t systemic txicity and nt as evidence f primary reprductive txic ptential. Als in 2002, the reprductive txicity f BPA was evaluated in a Eurpean Risk Assessment (published in 2003). It identified a need fr further research t reslve uncertainties surrunding the ptential fr BPA t prduce adverse effects n develpment at lw dses. Cnsequently, a 2-generatin study in mice accrding t OECD 416 (with sme specific mdificatins) was initiated. The muse study cnfirmed that BPA is nt a selective reprductive txicant. The study design and interpretatin f the results were supervised by a Steering Grup that was chaired by a representative f the Eurpean Chemicals Bureau and included experts frm several EU Member States; it was published as Tyl et al. (2008a). With the results f this 2-generatin muse study available, an updated Eurpean Risk Assessment f BPA was finalized in A weight f evidence review f the literature, including the 2-generatin muse study, cnfirmed the cnclusins f the 2002 EU Risk Assessment that BPA is nt a reprductive r develpmental risk t human health. The 2002 classificatin f BPA as Categry 3 remained unchanged as the new 2-generatin muse study cnfirmed that BPA is nt a selective reprductive txicant. Since 2008, additinal rbust and cmprehensive guideline type studies f regulatry relevance have been cncluded. These studies cnfirm that BPA is nt a selective reprductive txicant; fr example: In a study n develpmental neurtxicity (OECD 426) BPA was administered frm gestatin day 0 thrugh lactatin day 21 at dses up t 150 mg/kg bw/day: n effect n reprductive utcme were bserved (Stump et al. 2010). Mst imprtantly, very recently a new study cnducted at the US FDA Natinal Center fr Txiclgical Research (NCTR) became available (see Annex C fr detailed summary). In this study BPA was administered t rat dams by ral gavage frm gestatin day 6 until parturitin and then directly t pups frm pstnatal day 1 until terminatin at pstnatal day 90 at dses up t 300 mg/kg bw/day. BPA exhibited sme adverse effects at high dses (in Page 5 f 23

6 females at 100 and 300 mg/kg bw/day and in males at 300 mg/kg), and the authrs indicate in the study reprt that the interpretatin f the high dse BPA effects are cnfunded by systemic txicity. When the results frm studies with BPA are cmpared with the CLP Regulatin s requirements fr a substance t be classified as Categry 1B, it is clear that BPA shuld nt be classified as Categry 1B (see Annex D fr summary f relevant studies). The CLP Regulatin requires that: if, based n data frm animal studies, there is clear evidence f an adverse effect n sexual functin and fertility r n develpment frm expsure t the substance in the absence f ther txic effects, r if ccurring tgether with txic effects, the adverse effect n reprductin is cnsidered nt be secndary, nn-specific cnsequence f ther txic effects. Based n all the available scientific data, BPA des nt fulfil the criteria t qualify fr Categry 1B nr t be cnsidered a selective reprductive txicant: effects n animal fertility nly ccur at high BPA dses where already significant systemic txicity (e.g. reduced bdy weight f dams, effects n liver and kidneys) are seen there is n cnclusive evidence f adverse primary effects n sexual functin, fertility r develpment. In additin, based n cmprehensive multigeneratin reprductin txicity studies in rdents there is a clear gap between the verall N Adverse Effect Level fr general systemic txicity (5 mg/kg bw/day, ral dsing) and the dses at which bservatins n reprductin parameters were seen (500 mg/kg bw/day in rats and 600 mg/kg bw in mice, ral dsing; in the presence f systemic effects). In summary, BPA is nt a selective reprductive txicant based n guideline studies that cver a very wide dse range, frm very lw up t high dses. Secndary effects n animal fertility nly ccur at high dses f BPA that als shw clear evidence f systemic txicity. Cnsequently, the criteria fr classificatin f BPA as a Categry 1B Reprductive Txic are nt met. B. The CLH prpsal fails t engage in a weight f the evidence analysis as required by ECHA s Guidance The analysis underlying the CLH prpsal is nt cnsistent with the prcedure utlined in ECHA s Guidance n the preparatin f dssiers fr harmnised classificatin and labeling (ECHA 2010) because it fails t use a weight-f-evidence apprach apprpriate fr cmpunds, such as BPA, with a large database. 2 Specifically, the CLH prpsal dssier fails t fulfil criteria utlined in Sectin f Annex I t the CLP Regulatin EC1272/2008 (bld type highlight added) 3 : : A weight f evidence determinatin means that all available infrmatin bearing n the determinatin f hazard is cnsidered tgether, such as the results f suitable in vitr tests, relevant animal data, infrmatin frm the applicatin f the categry apprach (gruping, read-acrss), (Q)SAR results, 2 Sectin f Annex I t the CLP Regulatin and Sectin 1.2 f Annex XI t the REACH Regulatin 3 Page 6 f 23

7 human experience such as ccupatinal data and data frm accident databases, epidemilgical and clinical studies and well dcumented case reprts and bservatins. The quality and cnsistency f the data shall be given apprpriate weight. Infrmatin n substances r mixtures related t the substance r mixture being classified shall be cnsidered as apprpriate, as well as site f actin and mechanism r mde f actin study results. Bth psitive and negative results shall be assembled tgether in a single weight f evidence determinatin. 1. The CLH prpsal des nt cnsider all available infrmatin The time perid fr the literature included in the reprt is cnfusing due t incnsistent statements. 4 Hwever, what is clear is that the CLH prpsal mits relevant cmprehensive studies published in the literature befre December In particular it mits studies in BPA cnducted as part f a cmprehensive testing prgram by the US Natinal Center fr Txiclgical Research/FDA (e.g. Fischer et al. 2011, Derge et al. 2011a and b, Derge et al regarding txickinetics), as well as an OECD 426 Develpmental Neurtxicity Study (Stump et al. 2010). 2. The CLH prpsal des nt fllw the CLP Regulatin Guidance requirement that Bth psitive and negative results shall be assembled tgether in a single weight f evidence determinatin. The CLH prpsal discusses nly a limited and selected number f studies thrughut the reprt fr each endpint and des nt evaluate all psitive and negative results in a single weight f evidence determinatin. Fr example, the CLH prpsal failed t nte several studies reprted in the literature which investigated sperm parameter. The CLH Prpsal prvides n ratinale fr the inclusin/nn-inclusin f studies n this tpic. A literature review t simply identify sperm parameter studies revealed the fllwing studies that were nt cllected r analysed cnsidered in the CLH prpsal: Aikawa et al. 2004, Tyama et al. 2004, Kub et al 2003, Wistuba et al 2003, Pecnicva et al 2002, Park et al 2005b, Kub et al 2001, Cagen et al 1999, vm Saal et al 1998, Ashby et al 1999, Talsness et al 2000, Ema et al 2001 and mre recently Kendig et al. 2012, Xie et al. 2010, Zhang et al. 2012b. (Nte: In ur cmments cncerning the CLH prpsal we have nt evaluated these additinal studies fr quality, but include them as examples f available data that the CLH prpsal failed t cllect r analyse. See als Annex A fr further details and examples). A majr flaw in the CLH prpsal is its inclusin f studies in which the initial findings culd nt be reprduced, and its failure t mentin studies dne in the same labratry r in different labratries that demnstrated the nn-reprducibility f the riginal study. There are several cases where studies reprting effects are included, but repeat studies nt reprting the effect are nt included. Fr example: Hunt et al and Susjiarj et al CLH prpsal pages 64 and 114; tables 8 and 11. The CLH prpsal cites tw in viv studies (Hunt et al and Susiarj et al. 2007) fr the prpsitin that shrt-term ral expsure t lw dses f BPA in peripubertal r pregnant mice can interfere with meitic divisins in develpment f female germ cells ( egg r 4 Page 9 indicates: This prpsal is based n the studies presented in this French reprt (i.e. cnsidered by the French experts as key studies, irrespective f their publicatin date) tgether with all the new data published since 2002 n fertility (bibligraphical search stpped 31/12/2012). Hwever, page 117 refers t anther data range (exhaustive literature search frm 2002 t 2011). Page 7 f 23

8 cyte ). An increase in hyperplid (aneuplid) metaphase II cytes was bserved. There was nt a significant increase in aneuplid embrys. Nt included in the CLH prpsal are tw subsequent in viv studies (Pacchiertti et al. 2008, Eichenlaub-Ritter et al. 2008) that attempted unsuccessfully - t replicate these findings regarding aneuplidy. They detected n significant effects f BPA expsure n the frequency f aneuplidy in zygtes (fertilised cytes) prduced frm female mice treated befre puberty r as adults with a similar range f dses. In additin, Eichenlaub-Ritter et al. (2008) fund n effects f BPA expsure n aneuplid cytes and Pacchiertti et al. (2008) fund n increase in aneuplid r diplid sperm fllwing expsure f male mice t BPA. The authrs cncluded that the aneuplidy predicted by the Hunt grup culd nt be cnfirmed. This incnsistent picture was nted in the 2008 EU Risk Assessment. Als nt mentined in the CLH prpsal is a further study, Muhlhauser et al. 2009, which was published by the Hunt grup, in which the authrs culd nt replicate their wn initial findings n cngressin failure but reprt effects n chrmsme alignment and/r spindle frmatin. The authrs state: After publishing ur findings [Hunt et al., 2003], we initiated studies t assess the effect f lng term BPA expsure n the grwing fllicle. T ur surprise, levels f BPA that were sufficient t elicit an effect n meitic chrmsme dynamics during the previus tw years f study suddenly prduced little r n effect. In an analysis f pssible changes in experimental prtcl, the nly change identified was the lt f animal feed. (Muhlhauser et al. 2009, page 1066.) The authrs reprt frequencies f abnrmal cytes in the absence and presence f BPA in tw different diets (casein based and sy based). The reprted frequencies f abnrmal cytes f the BPA/casein grup are lwer than the backgrund value reprted in the sy-based diet. In cnclusin, the initial bservatins reprted by the Hunt labratry were nt reprduced in the same labratry r in ther independent labratries. The missin f this imprtant infrmatin frm the CLH prpsal fails t accurately represent the state f the science and abrgates the bligatin f the CLP Regulatin t assemble bth psitive and negative results in a single weight f evidence determinatin. Sakaue et al CLH prpsal page 92 and table 16 In Sakaue et al , the authrs have described hw ral expsure f sexually mature male rats t BPA between pstnatal days (PND) led t a reductin in daily sperm prductin (DSP) 5 weeks later (18 weeks f age). Activity was bserved ver the dse range 20 µg/kg 200 mg/kg BPA, with an absence f activity ver the dse range 2 ng/kg 2 µg/kg BPA. There was n evidence f a dse respnse relatinship ver the active dse range (five rders f magnitude range). But, the CLH prpsal des nt mentin an independent repeat f this study by Ashby et al which has been included in ther publicly available evaluatins f BPA, e.g. EFSA s Nvember 2006 pinin and CERHR s 2007 reprt, published in Birth Defects Research (2008). Ashby cnducted a ttal f fur independent studies accrding t the prtcl used by Sakaue et al (2001) and did see any evidence f changes in DSP reprted by Sakaue et al. If the CLH prpsal had evaluated bth studies in a weight f the evidence evaluatin, the cnclusin might be similar t CERHR s which stated: These data als strngly suggest that bisphenl A administered rally has n effect n sperm prductin albeit fllwing nly 6 days f administratin. 5 (Nte that the data given in table 16 n page 102 d nt crrespnd with the data given n page 92 f the CLH reprt r the riginal publicatin). Page 8 f 23

9 Myers et al Cmmentary CLH prpsal page 95 The CLH prpsal als references the Myers et al cmmentary criticising the Tyl el al. 2008a multi-generatin study, but des nt mentin r evaluate the published respnses by Dr. Tyl (Tyl 2009). The missin f Dr. Tyl s respnse is indicative f a pattern and practice in the CLH prpsal t selectively prvide nly data that supprts its prpsal. 3. The CLH prpsal fails t fllw the CLP Regulatin requirement that the cnsistency f the data shall be given apprpriate weight Incnsistency in data may indicate that an effect is either transitry, due t nrmal variatin r nt a repeatable finding. In many instances, the CLH prpsal fails t accunt fr incnsistencies in data. One example is given belw (fr further details see Annex A): On page 97, the CLH prpsal puts frward its Cnclusin n male reprductive system in animals, and states that: In the animals treated in uter and/r lactatin, mst f the studies perfrmed in mice r rats fund effects n sperm prductin r quality (Tinwell et al. (2002); Salian et al. (2009c)) But this statement is nt accurate because there are many additinal studies mentined in the CLH prpsal which reprt n effect n sperm, and the CLH prpsal itself recgnizes this fact n page 85 In cntrast t these previus studies, several authrs fund nly limited effect (Tinwell et al., 2002 and Kbayashi et al., 2010 and 2012) and ther failed t demnstrate significant effects f BPA expsure n the male reprductive tract, especially at lw dses (Hwdeshell et al., 2008; LaRcca et al., 2011). n page 86: It is difficult t find any specific reasn explaining thse cntradictry results with the state f actual knwledge. n page 88: All BPA grups in which analysis were perfrmed at PND10, 35 and 150 shwed nrmal reprductive parameters (fr instance preputial separatin, sperm analysis, serum teststerne levels, cpulatry and fertility rate, sexual rgan weight. Overall, the CLH prpsal fails t address incnsistency in the data t reach a weight f evidence determinatin as required by the CLP Regulatin. 4. The CLH prpsal fails t fllw the CLP Regulatin requiring that The quality f the data shall be given apprpriate weight. The CLH prpsal des nt define any quality criteria that it used t evaluate individual studies nr des it prvide any explicit criteria fr the inclusin r exclusin f studies frm the reprt. Rather than discuss the strengths and weaknesses f individual studies, the CLH prpsal merely repeats the data and cnclusins f the study authrs withut further discussin r evaluatin f the published data and withut cmparisn with ther available data, including histrical cntrl data and experience with the test system, and variability. Since n strength r weaknesses are indicated fr individual studies it is nt pssible t gauge the weight given in the CLH prpsal t individual studies. By nt evaluating the quality f the data and incrprating the data quality in the weight f the evidence analysis, the CLH prpsal fails t meet the basic requirements f ECHA s Guideline. Only indirect infrmatin n the criteria used in the CLH prpsal is available. The CLH prpsal des cite an ANSES interim reprt dated In this interim reprt a minimum f Page 9 f 23

10 six animals was cnsidered sufficient if the effect under investigatin displays high variability (hrmnal dse, number f sperm per ejaculate), a larger number f animals is necessary. The number f animals, but nt the variability f the endpint, is indicated in tables thrughut the CLH prpsal. But, there are many studies cited by the CLH prpsal that fail t meet the ANSES criteria f a minimum f six animals and are designated as N 5 r n data n the number f animals. Yet, the CLH prpsal des nt explain why these studies were cnsidered t be relevant, even thugh the ANSES interim reprt in 2011 cnsiders studies with N 5 as insufficient. By including, withut explanatin, studies that d nt meet its wn criteria fr data quality, the CLH prpsal apparently fails in its fundamental duty t rely nly n quality data. Befre relying n the data frm any study, the statistical pwer f an experimental design shuld be examined fr its ability t detect effects f a given magnitude. Fr example, in nenatal studies the litter shuld be the statistical unit f cmparisn, and nt the individual ffspring. This parameter was identified as an imprtant criterin fr study evaluatin by ther safety assessment panels, fr example CERHR 6 (NTP s Center Fr The Evaluatin f Risks T Human Reprductin) and by EFSA 7. In cntrast t the CLH prpsal, CERHR used defined criteria t evaluate many studies mentined in the CLH prpsal and cnsidered the studies as either inadequate r adequate but with limitatins. EFSA s 2008 Opinin fllwed a similar apprach which included ratinale, results, statistical issues, strengths and weaknesses f each study, and cnsequently the reasning fr EFSA t include r nt include the individual study int their assessment. Neither f these assessments nr their cncerns with the studies was mentined in the CLH prpsal. A detailed cmmentary cncerning the studies n which the CLH prpsal relies and which track the sectins f the CLH prpsal can be fund in Annex A. A brief summary f the CERHR Reprts assessment f studies mentined in the CLH prpsal in the chapter Summary and discussin f reprductive txicity is prvided here as an illustratin f the CLH prpsal s failure t cnsider study quality and the cnsequent reliance n inadequate r inapprpriate studies fr its prpsal: Aikawa et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate and nt useful based n small sample sizes and inadequate presentatin f statistical methds f analysis. Akingbemi et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: Experiment 2 is inadequate fr cnsideratin due t inapprpriate statistics that failed t accunt fr litter effects. Al-Hiyasat et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate fr the evaluatin based n small sample size. Berger et al. 2007: Utility (Adequacy) fr CERHR Evaluatin Prcess: Due t the absence f key infrmatin and faulty methdlgy, this study is inadequate fr evaluatin prcess. Chitra 2003: A weakness includes the marginal sample size. Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is adequate fr inclusin but f limited utility based n small grup size. 6 U.S. Department f Health and Human Services, Natinal Txiclgy Prgram, Center Fr The Evaluatin f Risks T Human Reprductin, NTP-CERHR Mngraph n the Ptential Human Reprductive and Develpmental Effects f Bisphenl A, September 2008, NIH Publicatin N , available n the web at: Page 10 f 23

11 Evans et al. 2004: The unique animal mdel and the use f LH pulsatile respnse are uncmmn but interesting. The high-dse level via i.m. injectin is a weakness as are small sample sizes (n 5 6). The statistical tests fr LH trends did nt appear t take int accunt the repeated nature f the sampling leading t ver stating the significance f trend effects. Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is adequate fr inclusin but f limited utility fr the evaluatin prcess. Herath et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate and nt useful fr the evaluatin prcess primarily due t the significant incnsistencies in the hrmne data frm cntrl animals. Hnma et al. 2002: The lack f AGD measurement at birth and difficulty f measurement at PND 60 are weaknesses. The Expert Panel was unable t cnfirm the statistical significance f the effects shwn in Table 2 f the manuscript. Utility (Adequacy) fr CERHR Evaluatin Prcess: The study is adequate fr inclusin but f limited utility due t statistical questins abut bdy weight and AGD and subcutaneus rute f expsure. Hwdeshell 1999: The missin f a descriptin f husbandry cnditins and lack f clarity f statistical prcedures are weaknesses. Use f nly a single dse is a weakness. Further, the use f time frm vaginal pening t first estrus is nt a standard endpint fr assessing puberty in mice and is f questinable bilgical significance. Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is adequate fr the evaluatin prcess but utility is limited due t uncertainties in data analyses. Iida et al. 2002: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate fr the evaluatin prcess based n methdlgy. Kabut et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate fr the evaluatin prcess due t inapprpriate statistical prcedures and small sample size. Nikaid et al. 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate fr the evaluatin prcess. Rubin et al. 2001: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate fr the evaluatin prcess, based n a lack f adequate cntrl fr litter effects Savabieasfahani et al. 2006: Weaknesses are the use f a single dse level and the relatively small sample size. The single time pint fr bisphenl A plasma determinatin at an unknwn time relative t s.c. injectin is a weakness. Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is adequate thugh f limited utility. Takahaski and Oshi 2003: Weaknesses include use f single high dses administered fr different duratins acrss grups using minimal sample sizes. Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is adequate but f limited utility. Tyama and Yuasa 2004: Utility (Adequacy) fr CERHR Evaluatin Prcess: This study is inadequate and nt useful due t critically small sample size, rute f administratin, lack f clarity f design, and inapprpriate statistical prcedures. Page 11 f 23

12 The CERHR Reprt s criticisms f fundamental study aspects, such as sample size, inapprpriate statistical prcedures, and even methdlgy, highlights the lack f quality in the studies relied n fr the cnclusins f the CLH prpsal. Its example f rbust quality analysis als highlights the failures f the CLH prpsal t meet the requirements f the CLP Regulatin t define quality criteria fr individual studies, evaluate studies fr quality, and perfrm a transparent weight-f-evidence evaluatin. 5. The infrmatin n reprductive endpints reprted in the CLH prpsal is incnsistent thrughut the prpsal In studies that investigate ptential reprtxic effects, the animal can be treated and/r mnitred fr effects in varius different stages f life: the dam, during gestatin, pregnancy immediately after giving birth, during lactatin, during mating and in the ffspring as nenatals, pups, in puberty, and during mating phase The perid f bservatin can be a few hurs up t several generatins f animals etc. The dses applied, the frequency, rute and duratin f applicatin add anther multiplying factr. In rder t avid cmparing apples with pears in data frm such a variety f study designs it is imprtant t be very transparent and specific in selecting and describing the endpints and related parameters that frm the basis f a reprt and related cnclusins. Hwever, thrughut the CLH prpsal, the allcatin f studies t the specific treatment intervals is incrrect and nt cnsistent. The scientific ratinale fr the chsen differentiatin f intervals is missing. N scientific reasning is given fr the exclusin f multigeneratin studies frm the assessment f defined intervals, althugh ther study results within the dsing scheme f the multigeneratin studies are included. OECD test guidelines as well as the glbally fllwed testing prtcls are based n the assumptin that a study result is relevant fr the interval f dsing that is cvered. In a study which cvered several dsing intervals (e.g. frm birth until next pregnancy) it is pssible that an adverse effect might be revealed. It might then be wrthwhile t further investigate the specific interval in which the adverse effect was induced. Hwever, in cases where a study with a large dsing interval des nt reveal an adverse effect, there is n need t examine specific intervals r t exclude the large interval studies frm the assessment f a specific interval. In a prper weight f evidence apprach, it is nt acceptable t exclude studies which shwed n effect and nly include studies that did shw an effect, neither is it apprpriate t exclude the results f such studies with respect t a specific interval nly because it shwed n effect. N effect is als a result. Hwever: this is the apprach the CLH prpsal takes. 6. Differences in strain sensitivity are mentined thrughut the CLH prpsal, but the statements are cntradictry, and relevant infrmatin n strain sensitivity is nt included. The CLH prpsal makes cntradictry statements regarding Sprague-Dawley (SD) rats: page 53: They are insensitive t endcrine mediated txicity page 60: have lw sensitivity t estrgenic cmpunds page 60: are fund respnders t estradil Page 12 f 23

13 Als with mice a statement n strain sensitivity is included in the CLH prpsal: C57BL/6N muse was estrgen-sensitive whereas ICR mice were fund insensitive (page 93) The prpsal als takes an incnsistent apprach t strain sensitivity in its summary tables: The prpsal summarizes effects n different endpints in tables, but included in general nly infrmatin n psitive findings (shwing an effect) and did nt indicate infrmatin n endpints with n bserved effect in these tables. There are several studies mentined in these tables which investigated SD rats r ICR mice. The fllwing studies in SD rats were cnsidered relevant in the CLH prpsal: Rubin et al. 2001, Takagi et al. 2004, Fernandez et al. 2009, Fernandez et al. 2010, Schönfelder et al. 2004, Tyl et al (page 53 f the CLH prpsal); whereas the fllwing studies were cnsidered nt relevant based n strain sensitivity: Ema et al. 2001, Tyl et al. 2002, Kwn et al There is a similar incnsistency fr studies in ICR mice. Althugh discussed as insensitive based n the Naga et al study the fllwing studies utilizing ICR mice which fund effects are cnsidered relevant by the CLH prpsal: Fernandez et al. 2010, Hiyama et al. 2011, Hnma et al. 2002, Nah et al. 2011, Nikaid et al Overall, the CLH prpsal des nt recncile hw strain sensitivity in SD rats r ICR mice is nt relevant when bservatins are reprted, but is relevant in n-effect studies using the same strains. Nr des the CLH prpsal recncile the missin f highly relevant infrmatin (e.g. EFSA 2010, CERHR 2008) and studies (e.g. Tyl et al. 2006, Gray et al. 2010, Tyl et al. 2008b and 2009) that address ptential species and strain differences; specifically: CERHR 2008 cnclusins f ptential differences in strain sensitivity: The differences in utcmes cannt be attributed t the use f an insensitive strain r stck because a variety f rat mdels were used in the negative studies: Sprague- Dawley, Wistar, Wistar-Furth rats, Wistar-derived Alderley Park, CD, and Dnryu. Mrever, three f the negative rat puberty studies reprted ther lw dse effects (53, 122, 173). The 2010 EFSA cnclusin n strain sensitivities, which is nt included in the CLH prpsal, said that: lw dse effects f BPA in rdents have nt been demnstrated with the sufficient certainty t serve as pivtal studies fr risk assessment. The mre recent bservatins f species differences in txickinetics f BPA between primates, including humans, and rdents, and in particular the lw biavailability f BPA (free systemic BPA) in primates, further weaken the relevance f bservatins f lw-dse effects f BPA in sensitive strains f rdents fr human health risk assessment. Gray et al. 2010, stated in a rebuttal t previus criticism: The LE and SD rat strains als are excellent animal mdels fr the study f the effects f EE2 and ther envirnmental estrgens because the sensitivity f these rat strains is very similar t the sensitivity f humans t EE2. The CLH prpsal als references the Myers et al cmmentary criticising the Tyl el al multi-generatin study, but des nt mentin r evaluate the published respnses by Dr. Tyl (Tyl 2009). Tyl cmmented as fllws n claims by Myers et al. 2009: We identified the same BPA systemic and reprductive/develpmental NOAELs (and sensitivity cmparable t similar dietary E2 intakes) in rats and mice, Page 13 f 23

14 with n BPA effects n the prstate weight r histpathlgy. Strain differences in respnse t estrgens in rats (and mice) vary acrss tissues, s n strain can be cnsidered mre sensitive than anther (Hwdeshell et al. 2008). E2 activities via estrgen receptr-α in the reprductive tract did nt display majr strain differences in OECD multilabratry rat utertrphic assay validatin studies; ral BPA was nly a weak partial agnist at mg/kg/day (Kann et al. 2003). Based n the additinal infrmatin and taking als int accunt the mst recent NCTR subchrnic txicity study in SD rats (which included EE2 psitive cntrl grups), the cntrary cnclusin is crrect: there is n slid scientific evidence that SD rats are insensitive t estrgenic cmpunds, and in general n strain can be cnsidered mre sensitive than anther. 7. The CLH prpsal inapprpriately rejects the results f guideline studies based n validated prtcls, yet accepts withut questin the results f explratry studies The statement in the CLH prpsal that data derived in guideline studies d nt cnfirm the data derived frm sme explratry studies is crrect. The lack f cnfrmity shuld raise questins abut the results f the nn-guideline studies, nt, as the CLH prpsal suggests, raise questins abut the results f the guideline studies. Guideline studies are cnducted accrding t methds that have been validated by repeated testing and fund t be reliable; they als specify sample size and ther parameters that cntribute t the reliability f the results. It is fr these reasns that guideline are bradly accepted as reliable methds. Therefre, the statements in the CLH prpsal The guideline studies cntradict the ther studies reprted withut straightfrward explanatin f this discrepancy (page 42) and The authrs d nt explain this difference. (page 65) inapprpriately reject withut explanatin results frm studies cnducted in accrdance with internatinally validated, apprved and accepted test guidelines. Indeed, it is the explratry studies with limited study design that require quality review t cnfirm that the study design, sample size and statistical pwer are sufficient fr their data t be cnsidered reliable, reprducible, rbust and relevant. The CLH prpsal, as discussed abve, has nt prvided a review f the quality f the explratry studies and many have been fund inadequate in prir gvernment reviews, such as CERHR, EFSA and the EU RAR. Thse same reviews by CERHR, EFSA and the EU RAR als reviewed the quality f the guideline studies and fund them t be f extremely high quality, reliable and cnsistent; in particular, acrss the guideline studies there was cnsistently n adverse effects n reprductive r develpmental endpints that wuld supprt a classificatin f BPA as categry 1B reprductive txicant. 8. Epidemilgical studies referenced in the CLH prpsal prvide n evidence t supprt stricter classificatin f BPA The epidemilgical studies referenced in the CLH prpsal t supprt its argumentatin are incapable f prviding any meaningful evidence f causatin because f their methdlgical apprach (crss-sectinal, single-spt sample measurement). In these studies, health effect and chemical expsure data are cllected at the same pint in time, which means there is n way t knw, based n the data evaluated, if the expsure preceded the disease. Withut this tempral infrmatin, statistical assciatins between expsure and health effects may be derived, but it is nt pssible t establish any causal relatinship between any bserved disease and BPA-expsure, because the respective infrmatin t assess such a relatinship is nt available in these studies. Page 14 f 23

15 Mst f the epidemilgical studies referenced in the CLH prpsal are f crss-sectinal design, and they base their finding n ne single bld r urine sample. Recent studies have shwn that, fr investigating substances with a shrt half-life, crss-sectinal studies, such as the US CDC s NHANES data set, are particularly inapprpriate (LaKind et al. 2012) 8. BPA has a very shrt half-life f nly a few hurs. Studies have shwn that BPA levels in urine are highly variable even within ne day. It is therefre impssible t draw cnclusins frm a single BPA measurement abut any ptential human disease which needs mnths r years t develp (e.g. Twnsend et al. 2013, Valvi et al. 2013, Philippat et al. 2013). Several f the authrs nte themselves that their findings need further investigatin, and a number f the studies display significant methdlgical flaws. Abut ne third f the included studies reprt n effect at all (see Annex E fr review f epidemilgical studies used in the prpsal). Overall, the crss-sectinal epidemilgical studies relied n in the CLH prpsal are inapprpriate t prvide any meaningful infrmatin as t ptential effects f BPA in humans. C. Statements abut the multigeneratin animal studies upn which regulatrs have relied (Tyl et al and 2008a) are incnsistent, incrrect and cntrary t evaluatins f these studies The Tyl et al. (2002 and 2008a) multi-generatinal studies f BPA in rdents are widely regarded as authritative research n BPA reprductive effects. These studies have been repeatedly relied upn by gvernmental regulatrs in assessing the risks and hazards f BPA. Tyl et al. (2002 and 2008a) have been credited fr their statistical pwer, wide range f dses and adherence t established guideline prtcls. The 2008 Eurpean Risk Assessment Reprt said: We cnsider this investigatin by Tyl et al. (2007) as the gld-standard, definitive study f the reprductive txicity f BPA (fr the endpints examined). 9 Likewise, the FAO/ WHO reprt said that: Typically, a dse f 5 mg/kg bw per day has been identified as a NOAEL in assessments cnducted fr regulatry r health-based guidance value setting purpses, based n cnsideratin f tw multigeneratin studies in rats and mice cnducted by Tyl et al. (2002, 2008a). These studies are generally cnsidered t be statistically and methdlgically sund fr the end-pints investigated and have sufficient dse grups t supprt dse respnse mdeling. 10 Tyl 2002 was described by NTP as arguably the mst cmprehensive f the studies we evaluated LaKind JS, Gdman M, Naiman DQ (2012) Use f NHANES Data t Link Chemical Expsures t Chrnic Diseases: A Cautinary Tale. PLS ONE 7(12):e Available at 9 Eurpean Cmmissin, Jint Research Centre, Institute fr Health and Cnsumer Prtectin, Updated Risk Assessment f 4, 4 Isprpylenedephenl (Bisphenl-A), Human Health Addendum, April 2008, p Fd and Agriculture Organizatin f the United Natins, the Wrld Health Organizatin, Jint Expert Meeting t Review Txiclgical and Health Aspects f Bisphenl A: Summary Reprt, Nvember 1-5, 2010, available n the web at: ftp://ftp.fa.rg/ag/agn/agns/bpa_summary_reprt.pdf, p Natinal Txiclgy Prgram, Reprt f the Endcrine Disruptrs Lw Dse Peer Review, page 1-11, August Page 15 f 23

16 The CLH prpsal mischaracterized imprtant elements f the multigeneratinal reprductive studies n rats (Tyl et al. 2002) and mice (Tyl et al. 2008a), as described in detail in Annex B and in summary belw. The CLH prpsal mistakenly claims that fertility effects were bserved in Tyl et al. 2008a when the study authrs did nt identify fertility effects frm the data. The interpretatin by the study authrs is supprted by the Eurpean RAR, which stated that: In the muse 2-generatin study, using dse levels f mg/kg/day, n effects n fertility, reprductive rgan weights and histpathlgy r sperm prductin were bserved. 12 The CLH prpsal likewise claims that BPA caused pituitary effects in Tyl et al. 2008a. Study data (discussed in Annex B) shw islated increases in pituitary weight and pituitary relative weight in males nly. These data pints d nt supprt a plausible dse respnse, and d nt suggest a treatment related effect. Neither the authrs nr the Eurpean RAR identify treatment related pituitary effects. The CLH prpsal cites Tyl et al. 2008a as supprting claims f extended estrus. The data d nt shw a clear pattern f extended estrus amng treated females. There were n statistically significant findings in the number f F0 r F1 females in estrus at any dse when cmpared t cntrls. The authrs cncluded that stage f estrus at demise was unaffected in mice. The CLH prpsal als claims that extended perids f diestrus were bserved in Tyl et al. 2008a. In fact, the percentage f F0 and F1 females in diestrus was ntably cnsistent acrss the range f dses. The data in Tyl et al. 2008a des nt supprt the CLH prpsal s statement n extended perid f diestrus. The CLH prpsal cntends that CD-1 mice and SD rats are insensitive t estrgenic cmpunds, suggesting this may accunt fr the lack f reprductive effects bserved in the Tyl studies. As discussed in Sectin B(6) f these Cmments (abve), the data d nt supprt strain in sensitivity. In fact, Tyl et al. (2008a and 2008b) included a cncurrent psitive cntrl f 17β-Estradil which clearly demnstrated the respnsiveness f this muse mdel t estrgenic substances. Similarly, the U.S. Fd and Drug Administratin sub-chrnic study n SD rats utilized a cncurrent psitive cntrl (EE2) which shwed clear indicatins f estrgenic respnse. The CLH prpsal s claims f estrgenic insensitivity f CD-1 mice and SD rats are refuted by substantial data as recgnized by CERHR (2008), EFSA (2010) and Gray et al The CLH prpsal characterizes the renal tubular degeneratin and chrnic hepatic inflammatin bserved in Tyl et al as strng and direct effect f BPA n these rgans. In fact, these effects, at the relatively high dses f 50 mg/kg/bw and 500 mg/kg/bw, were judged by the authr and by the Eurpean RAR t be prducts f systemic txicity. The CLH prpsal cntends that delayed puberty is bservable in Tyl et al at the 50 mg/kg/bw and 500 mg/kg/bw dses. This statement is nt crrect. Tyl et al. 2002, Fig 7 shws n significant effect at 50 mg/kg. The CLH prpsal claims that effects n sperm cncentratin and accessry sex rgans ccurred in Tyl et al Because the data n sperm are nt cnsistent acrss the generatins, they were nt cnsidered by the study authrs t be treatment related. Further, Tyl et al fund n treatment-related grss r 12 Eurpean Cmmissin, Jint Research Centre, Institute fr Health and Cnsumer Prtectin, Updated Risk Assessment f 4, 4 Isprpylenedephenl (Bisphenl-A), Human Health Addendum, April 2008, p Page 16 f 23

17 micrscpic findings n reprductive rgans fr F0, F1, F2, r F3 adult males r females. The criticisms in the CLH prpsal abut these multigeneratin studies are neither supprted by the study data themselves, nr by ther independent reviewers such as the EU RAR r the US-CERHR Reprt bth f which cnfirmed the high quality f the design f the Tyl studies and the validity f their results. The Tyl studies d nt prvide a basis fr cncluding that BPA is a selective reprductive txicant and d nt supprt a classificatin f BPA as categry 1B reprductive txicant. D. Recent and imprtant infrmatin available n a new study cnducted at the US FDA Natinal Center fr Txiclgical Research (NCTR) is nt included in the CLH prpsal Very recently, findings frm a new study cnducted at the US FDA Natinal Center fr Txiclgical Research (NCTR) became available (see Annex C fr further details). In this study BPA was administered t rat dams by ral gavage frm gestatin day 6 until parturitin and then directly t pups frm pstnatal day 1 until terminatin at pstnatal day 90 at dses up t 300 mg/kg bw/day. In this rbust study, which features a cmprehensive range f dse levels and wide array f health endpints, BPA exhibited sme adverse effects in females nly at the high dse levels f 100 and 300 mg/kg bw/day and in males at 300 mg/kg. The authrs nte that the interpretatin f the high dse BPA effects are cnfunded by systemic txicity. Thus, the study supprts earlier research and Eurpean Risk Assessments cncluding that BPA prduces adverse effects nly at high dses assciated with systemic txicity. Overall the data in this cmprehensive study d nt supprt the premise that BPA is a selective reprductive txicant. This recent research further cnfirms that mre stringent classificatin f BPA as Categry 1B is nt warranted. E. The CLH prpsal is nt cmprehensive in cnsidering the available txickinetics data The CLH prpsal is based n an interim reprt by ANSES dated In that reprt ANSES indicated: A detailed analysis f the txickinetic data f BPA by species and rute f expsure is in prgress with a view t establishing crrelatins between the varius studies and the expsure levels in humans. The nw available CLH prpsal des nt mentin such a human PB-PK mdel (species-specific physilgy-based pharmac-kinetik), but nevertheless justifies the inclusin f many studies if nt the majrity - using the parenteral rute f administratin, thereby disregarding rute-dependent pharmackinetics f BPA. It is well established that the primary rute f expsure t BPA is ral. 13 Als well characterized is the txickinetic prfile f BPA (i.e. the fate f the substance in the bdy: its absrptin, distributin, metablisatin, eliminatin) in mice, rats, mnkey and human vlunteers. In humans and ther primates, multiple studies shw that rally ingested BPA is rapidly transfrmed t BPA-glucurnide during first pass metablism in the gut wall and the liver and that BPA-glucurnide des nt have any endcrine activity. Bth BPA and BPAglucurnide are rapidly excreted via the urine, with an eliminatin half-life f less than 6 hurs. Thus, there is very lw biavailability f the parent substance, BPA, in humans and ther primates (EU Risk Assessment 2008, Vlkel et al and 2005, Teeguarden et al., 2011 and 2013). 13 (US FDA and EFSA 2006a, 2008; EFSA Panel 424 n Fd Cntact Materials, Enzymes, Flavurings and Prcessing Aids (CEF), 2010, as well as DRAFT Scientific Opinin n the risks t public health related t the 4 presence f bisphenl A (BPA) in fdstuffs Part: expsure assessment (2013) Page 17 f 23

18 In humans, due t rapid bitransfrmatin, excretin and plasma prtein binding, peak BPAcncentratins after dietary expsures t BPA are predicted t be very lw even in wrst case expsure scenaris. In rats, rally administered BPA als predminantly underges glucurnidatin, but the BPA-glucurnide is excreted frm the liver int the gut in the bile. In the gut, BPA-glucurnide is then cleared int BPA and glucurnic acid and BPA is reabsrbed as such int the bld stream. This enterhepatic recirculatin results in slwer eliminatin f BPA in rdents (BPA half-life in rdents 19-78h) (EFSA 2006, Hengstler et al. 2011). Nn-human primates internal expsure t BPA is remarkably similar frm birth thrugh adulthd and cnsistently lwer than rdents during the nenatal phase. The results f the recent NCTR wrk indicates that if BPA dses causing adverse effects in rdent mdels were attributable t discrete nenatal develpment windws, such effects shuld be less likely fr cmparable nenatal primate expsures n the basis f internal dsimetry. (EU Risk Assessment 2008, Hengstler et al. 2011, Derge et al. 2010a and b). Txickinetic infrmatin is available fr ral and parenteral dsing in rdents and mnkeys frm multiple recent studies by the US FDA/NCTR 14.The data n txickinetics and metablism revealed substantially lwer internal expsure t BPA after ral dsing cmpared t parenteral dsing fr rat and mnkeys at all time pints investigated (newbrn t adulthd) and in mice frm juvenile t adulthd. Based n these data it can be cncluded, that parenteral rutes f administratin as s.c, i.m. injectin r smtic pumps will lead t substantially higher internal dses f BPA. In particular high parenteral dses f BPA shuld be regarded as unreliable based n EU Regulatin 1272/2008 chapter : Studies invlving rutes f administratin such as intravenus r intraperitneal injectin, which result in expsure f the reprductive rgans t unrealistically high levels f the test substance,, must be interpreted with extreme cautin and n their wn are nt nrmally the basis fr classificatin. As a review and analysis f the studies shw, BPA expsure is primarily ral and it is rapidly metablized in the gut which means that BPA-cncentratins after dietary expsures t BPA are predicted t be very lw even in wrst case expsure scenaris. In light f the txickinetic prfile, the CLH prpsal s heavy reliance n studies with parenteral rutes f expsure leads t unrealistically high levels f expsure which is nt an apprpriate basis fr classificatin. F. Reference t baby bttles as surce f expsure is utdated On page 23, with reference t an ANSES reprt f 2010, the CLH prpsal mentins plycarbnate bttle feeding and/r infant frmula feeding as a surce f direct infant expsure t BPA. Such expsure is n lnger existing, as since 1 June 2011, the sale f BPA-based plycarbnate baby bttles is n lnger permitted under Eurpean law. The decisin was taken after the market fr plycarbnate baby bttles in Eurpe had virtually disappeared. This decisin was nt a cnsequence f any evidence f adverse effects frm BPA, but rather a highly precautinary apprach by the EU. Nt ne 14 Derge et al., 2010a, 2010 b, 2010 c, Twaddle et al., 2010; Derge et al., 2011; Fisher et al.; 2011, Derge et al, 2012, Yang et al., 2013, Pattersn et al., Page 18 f 23