GM Free Cymru

Roundup and birth defects: Carrasco vs Monsanto

Carrasco sees off GM industry heavies.....

Sunday, 27 March 2011 18:46
http://www.gmwatch.org/index.php?option=com_content&view=article&id=13001

It was inevitable that the GM industry would go after Andres Carrasco and his colleagues, who had the temerity to demonstrate that glyphosate and Roundup are harmful. Of course it's harmful -- the stuff is after all an extremely toxic compound. It's designed to kill things -- and any small child would understand that it might well kill things other than those which it is intended to target......... and the idea that it is a selective weedkiller is utter nonsense. As usual, the scientists from Monsanto and Dow Chemicals try to muddy the waters, citing carefully selected references and falling back on their own industry studies and on supposedly comprehensive reviews of safety by assorted regulators across the globe. Carrasco is having none of it -- and goes after these guys with a vengeance......... and of course he has a vast (and growing) literature on his side.

1. Letter to journal editor from Monsanto regarding Carrasco's research

2. Response to Monsanto by Carrasco

NOTE: The study that is being discussed here, by a team led by Prof Andres Carrasco, found that glyphosate and Roundup cause birth defects in frog and chicken embryos at extremely low doses. It can be accessed here:
http://www.gmwatch.eu/images/pdf/Carrasco_research_paper.pdf

1. Letter to the Editor Regarding Paganelli et al. Article
Chem. Res. Toxicol., Article ASAP
Publication Date (Web): March 21, 2011
Copyright © 2011 American Chemical Society
http://pubs.acs.org/doi/abs/10.1021/tx100452k
 
To the Editor: Regarding the recent article by Paganelli et al. (Chem. Res. Toxicol., 2010, 23, 1586–1595) "Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling", we write to a) confirm the high degree of confidence in the substantial toxicological data base for glyphosate; b) discuss the unsubstantiated basis provided by the authors as rationale for this published research; and c) provide context for the dosing levels evaluated by the authors with respect to human health risk assessment.

a) Multiple high quality toxicological studies and expert review panels consistently agree glyphosate is not a teratogen or reproductive toxicant: The GLP studies that Paganelli et al. infer as untrustworthy "industry-funded studies" have been exhaustively reviewed by multiple government scientific regulators, often comprised of academic expert scientists and all of which have strongly supported the conclusions put forth in those studies. Glyphosate does not cause adverse reproductive effects in adult animals or birth defects in offspring of these adults exposed to glyphosate, even at very high doses. These conclusions are based on multiple studies in laboratory animals that have been conducted to examine the potential for multigenerational and teratogenic effects. These studies have been repeated by different companies at different laboratories across the globe over the last thirty or more years, with consistent results demonstrating that glyphosate does not pose the concerns raised by the authors. Regulatory authorities and independent experts who have documented this position include WHO/FAO (1), U.S. EPA (2), the European Commission (3) and Williams et al. (4).

b) Flawed Premise: The authors provide no valid basis, other than an opinion, of an increase in the rate of birth defects in Argentina. The referenced epidemiology paper (5) implied by the authors as justification for implicating glyphosate as a chemical of concern, does not mention glyphosate or even distinguish between herbicide, insecticide, molluscicide, rodenticide or fungicide potential exposures to pregnant women. This small epidemiological study, conducted in Paraguay, investigated associations between proximity or assumed exposure to pesticide use / storage and congenital malformations in neonates. The association between "living near treated fields" (distance and pesticide types unspecified) and congenital malformations was weak, with an odds ratio about six times lower than the reported association between pesticide storage in the home and congenital malformations. There is nothing unusual about the wide variety of birth defects reported in the Paraguay study and it provides no support for the authors’ allegation that they "strikingly resemble the wide spectrum phenotypes resulting from a dysfunctional RA or Shh signaling pathway".

The authors cite a number of papers (6, 7, 8, 9) suggesting glyphosate or glyphosate based formulations are cause for concern regarding endocrine disruption or human reproduction and development. These studies were all based on unvalidated in vitro test systems. Such methods, and some of the specifically referenced literature, have been reviewed by regulatory authorities around the world (3, 4, 10, 11, 12, 13) and other expert panels (14) and were consistently deemed inappropriate and irrelevant for human health risk assessment purposes.

c) Irrelevant routes of exposure and inappropriately high doses: The research described by Paganelli et al., 2010, exposed two-cell frog embryos via direct injections of 360 pg and 500 pg glyphosate acid per cell, bypassing the developing amphibian protective gel coat. Assuming a cell diameter of 1mm to determine spherical volume, the cellular doses are approximately 690 to 950 μg/L within each treated cell. Frog embryos were also bathed in glyphosate formulation at 1/5000 to 1/3000 dilutions of glyphosate formulated product (approximately 70000 μg/L to 120000 μg/L glyphosate respectively). These doses are 9-15 times greater than the acute LC50 value of 7900 μg/L for frog embryos of same species (15). Fertilized chicken eggs were also exposed via an unrealistic scenario, by opening a window in the shell and directly dosing 20 μL of 1/3500 and 1/4500 dilutions of glyphosate formulated product (2.0 and 1.6 μg/chicken embryo). Using a similar chick embryo assay, Kobayashi et al., (16) found the commonly consumed substance caffeine, to cause malformations in chick embryos.

A recent pharmacokinetic study in rats (17), found that a 400 mg/kg oral dose of glyphosate resulted in blood Cmax concentration of 4.6 μg/mL. Assuming linear pharmacokinetic behavior in rats for glyphosate, the dose necessary to produce a blood concentration of 72 μg/mL (as in the "low dose" of 72000 μg/L in the frog embryo culture experiments) in rats would be over 6200 mg/kg body weight (72 μg/mL / 4.6 μg/mL x 400 mg/kg body weight = 6261 mg/kg body weight). Thus, the in vitro concentration used by the authors was equivalent to a glyphosate oral dose to rats of 6261 mg/kg body weight. This dose is over an order of magnitude greater than the already high doses of glyphosate shown not to cause developmental or reproductive effects in rats and rabbits (NOAELs), which are used for risk assessment purposes by some regulatory authorities to establish safe human allowable daily intakes (ADIs).

Based on the findings from their report, the authors express their concern for "families living a few meters from where the herbicides are regularly sprayed". This exposure scenario of concern is similar to that directly evaluated in the Farm Family Study (18) in which spouses were biomonitored for glyphosate exposure during a period of intense spraying of the herbicide only a few yards from their homes. Yet, even with that exposure proximity, the maximum systemic dose to spouses in the Farm Family Exposure Study was 0.04 μg/kg body weight, with more than 95% of the spouse exposures below the limit of detection. The margin of exposure of this human biomonitored-measured dose relative to the rat equivalent dose used in the frog embryo bathing experiments exceeded 150,000,000 (rat equivalent dose of 6,261 mg/kg equals 72 ug/mL in frog embryos; MOE = 6,261,000 ug/kg/[0.04 ug/kg human dose] = 156,525,000). The rat equivalent dose is the appropriate comparator to develop the Margin of Exposure calculation in that mammalian toxicology studies are the primary datasets to assess human exposure risks, and indicates that the frog embryo in vitro doses used in this study were exceedingly unrealistic relative to potential human exposures resulting from field use of glyphosate.

In conclusion, the model systems employed by Paganelli et al., 2010, in which materials are tested at unrealistically high doses, may offer interesting results that help screen for early-tier toxicological effects, and perhaps offer some utility in elucidating hypothesized toxicological mechanisms. However, the results from this research cannot be used in isolation to reach the conclusions expressed in the publication. Instead, the type of data in this research paper must be interpreted relative to all other available data on the specific materials under study and with balanced consideration for higher tier apical studies. When all data including the extensive in vivo toxicological data base are evaluated together in this manner, the weight of evidence supports the corroborated conclusion of regulatory experts across the globe, that glyphosate is not a developmental or reproductive toxicant.

References

(1) WHO/FAO. (2004) Pesticides residues in food -- 2004. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticide Residues (JMPR). Rome, Italy, 20–29 September 2004. FAO Plant Production And Protection Paper 178. World Health Organization and Food and Agriculture Organization of the United Nations. Rome, Italy.
http://www.fao.org/ag/agp/agpp/Pesticid/JMPR/DOWNLOAD/2004_rep/report2004jmpr.pdf
(2) U.S. EPA. (1993) Reregistration Eligibility Decision (RED) for Glyphosate. U.S. Environmental Protection Agency, Office of Pesticide Programs. EPA 738-R-93-014.
http://www.epa.gov/oppsrrd1/REDs/old_reds/glyphosate.pdf
(3) European Commission. (2002) Report for the Active Substance Glyphosate, Directive 6511/VI/99, January 21. http://ec.europa.eu/food/fs/sfp/ph_ps/pro/eva/existing/list1_glyphosate_en.pdf
(4) Williams G, Kroes R, Munro IC. (2000) Safety evaluation and risk assessment of the herbicide Roundup® and its active ingredient, glyphosate, for humans. Regulatory Toxicology and Pharmacology 31: 117-165. doi:10.1006/rtph.1999.1371
(5) Benıtez Leite, S., Macchi, M. A., and Acosta, M. (2009) Malformaciones Congenitas asociadas a agrotoxicos. Arch. Pediatr. Drug 80, 237–247.
(6) Richard, S., Moslemi, S., Sipahutar, H., Benachour, N., and Seralini, G. E. (2005) Differential effects of glyphosate and roundup on human placental cells and aromatase. Environ. Health Perspect. 113, 716–720.
(7) Marc, J., Mulner-Lorillon, O., Boulben, S., Hureau, D., Durand, G., and Belle, R. (2002) Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation. Chem. Res. Toxicol. 15, 326–331.
(8) Benachour, N., and Seralini, G. E. (2009) Glyphosate formulations induce apoptosis and necrosis in human umbilical, embryonic, and placental cells. Chem. Res. Toxicol. 22, 97–105.
(9) Gasnier, C., Dumont, C., Benachour, N., Clair, E., Chagnon, M. C., and Seralini, G. E. (2009) Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology 262, 184–191.
(10) Afssa. (2009) Avis de l'Agence française de sécurité sanitaire des aliments relatif au glyphosate et aux préparations phytopharmaceutiques à base de cette substance active. Afssa – saisine n°2008-SA-0034 – Glyphosate. le 26 mars 2009. http://www.afssa.fr/Documents/DIVE2008sa0034.pdf
(11) Australian Pesticides and Veterinary Medicine Authority, APVMA (2010), Glyphosate is being reviewed in the United States and Canada. Is it still safe to use? 31 August, 2010. http://www.apvma.gov.au/news_media/community/2010-13_glyphosate_au.php
(12) Health Canada, Information Note: Request for a Special Review of Glyphosate Herbicides Containing Polyethoxylated Tallowamine. http://www.hc-sc.gc.ca/cps-spc/pubs/pest/_fact-fiche/glyphosate/index-eng.php
(13) U.S. EPA (2009). Public comments regarding the Health Effects Division’s (HED’s) human health assessment scoping document in support of registration reiview of 3-Jun-2009. HED’s response to public comments. EPA-HQ-OPP-2009-0361-0041. http://www.regulations.gov/search/Regs/home.html#docketDetail?R=EPA-HQ-OPP-2009-0361
(14) ECETOC TECHNICAL REPORT No. 106 Guidance on Identifying Endocrine Disrupting Effects, ISSN-0773-8072-106, Brussels, June 2009.
(15) Edginton A. N., Sheridan P. M., Stephenson G. R., Thompson D. G. and Boermans H. J. (2004) Comparative Effects of pH and Vision® Herbicide on Two Life Stages of Four Anuran Amphibian Species. Environ. Toxicol. Chem. 23(4): 815-822.
(16) Kobayashi T., Nishida A., Kurokawa A. and Fumio A. (1995). Cardiovascular malformations induced by caffeine amd Phenobarbital in chick embryos, AATEX 3, 17-27.
(17) Anadóna, A., Martínez-Larranaga, M. R., Martínez, M. A., Castellano, V. J., Martínez M., Martin, M. T., Nozal, M. J.and Bernal, J. L. Toxicokinetics of glyphosate and its metabolite aminomethyl phosphonic acid in rats Tox. Lett. 190: 91-95, 2009.
(18) Acquavella J. F., Alexander B. H., Mandel J. S., Gustin C., Baker B., Chapman P. and Bleeke M. (2004) Glyphosate biomonitoring for farmer-applicators and their families: results from the Farm Family Exposure Study. Environ. Health Perspect. 112:321–326.

David Saltmiras, Ph.D., DABT
Toxicology Manager
Monsanto Company
James S. Bus, Ph.D., DABT, ATS
Ph.D, DABT, Fellow, ATS
Director External Technology, Toxicology and Environmental Research and Consulting
The Dow Chemical Company
Terri Spanogle, B.S.
Toxicologist

2. Reply to the Letter to the Editor Regarding Our Article (Paganelli et al., 2010)
Andres E. Carrasco
Chem. Res. Toxicol.
Publication Date (Web): March 23, 2011
Copyright © 2011 American Chemical Society
http://pubs.acs.org/journal/crtoec

To the Editor: The letter to the editor sent by representatives of Monsanto, Syngenta, and Dow Chemicals (among others) to Chem. Res. Toxicol. regarding our paper (Paganelli et al., published July 23, 2010) and the tone used in their criticism about other research papers studying glyphosate effects should come as no surprise considering the obvious conflicts of interest inherent in this work when the companies selling a product are also solely responsible for testing its safety.

These multinational corporations handle virtually all of the seed and chemical products market in the world; therefore, it cannot be inferred that research performed or supported by such companies is completely objective. Their dismissal of our research and that of other researchers harkens back to the ongoing debate about bisphenol A, where no single industry funded study has ever found adverse consequences linked with BPA exposure, whereas 90% (n > 100) of nonindustry funded studies show significant adverse consequences of BPA exposure.1,2 Therefore, we contend that rather than pointing out shortcomings of our research, the letter illustrates the increasing difficulty in dialogues between those with a vested interest in product sales and independent researchers who wish simply to understand whether the said products are safe. It is worse yet when multinational corporations attempt to use their own flawed science to hide and defend environmental devastation suffered by less-developed countries in areas where their products are heavily used with only minimal governmental scrutiny.

POINT A
The objections to the effects of glyphosate noted by the authors clearly indicate that they did not review the complete evidence previously published by independent (i.e., non-industry funded) groups, who reported teratogenic effects equivalent to those reported in our work, Lajmanovich3 and Dallegrove,4 nor did they take note of the inhibitory activity produced by atrazine5 and glyphosate6 on CYP19 (cytochrome P450 aromatase) that affected sex steroid metabolism leading to endocrine disruption.

Furthermore, they ignore evidence that Triadimefon,7 glyphosate (our paper), and probably atrazine8 produce teratogenic effects through the alteration of the retinoic acid pathway. The claim that glyphosate is not teratogenic and does not produce adverse reproductive effects is based on studies generated by the industry, as the authors of the letter indeed recognize.

This constitutes a strong and material conflict of interest in the outcome of said studies. Moreover, these technical reports are often adopted as criteria for use by the state control agencies, without being corroborated experimentally by independent scientists. In fact, the WHO, in the 2009 document Classification of Pesticides Recommended by the WHO by Hazard and Guidelines to Classification (CPRHGC), states the following:

"All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use."

In a different paragraph of the same document, the WHO adds the following:

"Any classification based on biological data can never be treated as final. In the assessment of biological data, honest differences of opinion are inevitable and most borderline cases can be reclassified in an adjacent class. Variability or inconsistency in toxicity data due to differences in susceptibility of test animals, or to experimental techniques and materials used can also result in differing assessments."

Moreover, in the following paragraph, the WHO rescued a text from the proposal approved by the World Health Assembly in 1975:

"The classification criteria are guide-points intended to supplement but never to substitute for special knowledge, sound clinical judgment or experience with a compound. Reappraisal might be necessary from time to time."

In addition, the reports referenced by WHO (1994) are mainly based on technical information provided by companies interested in producing and marketing the product and its formulations. For example, 180 reports were performed and/or supplied by Monsanto. Among them, more than 150 were not published and therefore were not subjected to peer review. Other key technical reports provided as references in the same document, such as 17 reports from Agrichem (producer and marketer of pesticides based in The Netherlands), 5 from Luxan BV, (Netherlands), and 5 from Rhone Poulenc were not published either.

These reports are used in a complementary manner as the main source for the considerations used for classification. For example, the unpublished 1990 Monsanto report is quoted twice to justify the spraying of glyphosate, and the unpublished 1988 Monsanto report is quoted twice in reference to methodological aspects and metabolic transformations.

However, there are no reports concerning procedures for designating the teams responsible for the selection of studies used as reference, analysis, evaluation, and classification or the final considerations. In particular, there is no evidence provided that the people responsible for such reports have the required expertise or scientific oversight to make the reports credible. In the 2009 CPRHGC document, the WHO states the following:

"In practice, the majority of classifications will be made on the acute oral LD50 value. However, dermal toxicity must always be considered since it has been found that, under most conditions of handling pesticides, a high proportion of the total exposure is dermal."

This implies other reasons for considering the WHO classification insufficient to protect the health of the population and the environment from damage produced by agrochemicals. For instance, the classification is based primarily on acute oral and, eventually, dermal toxicity. While these determinations are standard procedures in toxicology, they leave out chronic toxicity and sublethal toxicity assays and do not consider access through the respiratory tract.

In addition, the 2004 report from the JMPR FAO/WHO Expert Meeting, often misleadingly cited as a scientific report by advocates of glyphosate, contains no scientific references that support the conclusions drawn within its 383 pages. It is unclear as to who performed the research, what methodology was used, and what form of peer review was employed to evaluate the quality of the material contained in the report. As a matter of fact, the document acknowledges in the Introduction that

"Most of the summaries and evaluations contained in this report are based on unpublished private property before the Committee to make assessments."

Therefore, we argue that it is high time that data used to determine whether products are safe are generated by independent entities with no ties to the manufacturers of the said products, or those who have a financial interest in the approval of the said products.

POINT B
The letter also criticizes the literature cited in our work. Benitez-Leite’s paper9 is a study that points to the correlation between malformations and exposure to pesticides in Paraguay, giving a precise idea of the outcome of heavy agrochemical use in Paraguay. The mentioned paper identifies living near treated soy fields, dwellings located less than 1 km from treated fields, storage of pesticides in the home, and contact with pesticides as significantly associated risk factors for congenital malformations.

This study brings concerns about the situation created in Paraguay by the expansion of industrialized soy crops which require the intensive use of agrochemicals (which, needless to say, includes glyphosate).

Despite the dismissal by the companies, several malformations observed by Benitez-Leite (for example, anencephaly, microcephaly, facial defects, myelomeningocele, cleft palate, synotia, polydactily, and syndactily) are indeed consistent with the well known and expected syndrome caused by misregulation of the retinoic acid (RA) pathway. RA is a well-known teratogen that causes craniofacial abnormalities (by misregulation of sonic hedgehog and otx2 expression) and posterior regression syndrome in all vertebrates tested including, unfortunately, humans.10,11 Notwithstanding the corporate disinformation provided by our critics, the malformations observed by Benitez-Leite et al. are indeed consistent with the well-known and expected malformations caused by increase of RA.

These conclusions should be taken into account together with studies on the incidence of malformations and cancer conducted in Chaco, an Argentine province with records in soybean harvest and use of glyphosate. These official records (often hidden by the Argentine government) reveal a 3-fold increase in developmental malformations in the province and a 4-fold increase cancer in the locality of La Leonesa.12

These data should be sufficient to raise the alert worldwide and lead to the commissioning of an independent study to provide an unbiased and dispassionate evaluation of the information rather than relying on studies commissioned by companies 13 or requested by the U.S. Drug Enforcement Agency in support of their efforts to eradicate coca plantations. Suggestively, an epidemiological surveillance conducted between December, 2004 and April, 2008 in Cali and Valle del Cauca in Colombia revealed that cyclopy is an endemic event with a prevalence 14- to 43-fold higher than that reported in the literature.14

Long before our work, reports appeared in the mainstream press about the effects of agrochemicals on human and animal health based on direct observations from physicians and health workers. This was a very important warning about the environmental consequences of using 200 million liters of GBH/year in Argentina and led to a vigorous debate about the safety of GBH and the "precautionary principle", urging to initiate epidemiological studies.15

Finally, we find unfortunate the expression "consistently inappropriate deemed irrelevant for human health and Risk Assessment purpose" with regard to the work of Dr. Seralini and Dr. Marc. This criticism is an unscientific value judgment that does not refute the quality of the work but rather seeks to discredit, without providing evidence to the contrary. Such comments are unwarranted in the scientific literature and should be considered together with the source: a corporate entity seeking to continue the production and use of a product that independent scientists have found to have adverse health consequences.

POINT C
Glyphosate penetration through the cell membrane and subsequent intracellular action is greatly facilitated by adjuvants such as surfactants.16,17 The authors of the letter conveniently avoid discussing this fact. Moreover, the companies they represent are not required to reveal the composition or safety of adjuvants used in the commercial formulations which are protected as trade secrets. It was for precisely this reason that we tested both the active principle, glyphosate, as well as commercial formulations for teratogenicity. This is a more realistic test of whether glyphosate or the formulations (which vary by manufacturer and intended use) is responsible for the malformations observed.

The calculated intracellular concentration for glyphosate injected into embryos was 60 times lower than the glyphosate concentration present in the 1/5000 dilution of the GBH which was used to culture whole embryos. Notwithstanding this, both gave similar phenotypes and changes in gene expression, suggesting that the effects are attributable to the active principle of the herbicide.

The authors of the letter claim that the glyphosate doses used are 9-15 times greater than the acute LD50 value for frog embryos of the same species. In fact, the study by Edginton et al. (2004) on which they support this statement uses a different commercial formulation. Because the adjuvant composition can change the permeability of the membranes to the active component, the effective concentration in the cells cannot be compared between studies. Moreover, results from Seralini’s group suggest that the adjuvants per se may pose adverse effects in cell cultures.18 Indeed, Edginton et al. claim that the surfactant is the major toxic component of the formulation they use. On the other hand, the LD50 of teratogens may vary between batches of embryos. The LD50 is not an accurate criterion to analyze the developmental defects that indeed occur in the survivors.

The authors representing the companies present a series of calculations according to the toxicological point of view, while cell biology and molecular and developmental biology are absent in their considerations. Moreover, they do not discuss the following key issues:

(a) In the field, usually the main route of systemic entry is the respiratory tract instead of the digestive tract.
(b) Direct blood concentration is only an average indicator of the presence of the chemical and does not provide evidence about its tissue distribution. Recently, a 2-compartment model study suggested that a considerable diffusion of the herbicide into the tissue is reached after intravenous administration.19
(c) They do not consider the fact that the human placenta is permeable to glyphosate; 15% of administered glyphosate by perfusion in vitro experiments trespasses the human placentary barrier.20
(d) They ignore the possibility that very low concentrations (pg/cell and not necessarily evenly distributed to all cells) may be sufficient to cause embryonic lethality (which is consistent with increased frequency of embryonic death and spontaneous abortions) or to modify normal embryonic pattern formation.
(e) They do not consider the paper of Dallegrove et al.4 who observed craniofacial ossification defects and loss of caudal vertebrae in rats orally treated with sublethal doses of GBH. These alterations were statistically significant (p < 0.05, χ2 test) in comparison with the control group and, importantly, were dose-dependent, indicating a specific effect.

Although these authors do not address the molecular basis of the teratogenic effects they observe, an altered retinoid signaling pathway is a major candidate to be considered, for the following reasons: normal craniofacial morphology is the result of complex interactions between embryonic tissues and requires precise regulation of cell movement, growth, patterning, and differentiation. Mutations or misregulation of genes that influence any of these processes would cause craniofacial abnormalities, such as facial clefting and craniosynostosis. Among the critical genes involved in craniofacial development is the Msx family of homeodomain transcription factors.21 Msx genes contribute in maintaining the balance between proliferation and differentiation during pre- and postnatal skull morphogenesis.

Mutant mice for msx2 show incomplete or delayed ossification of the calvarial bones (i.e., those that constitute the upper part of the cranium and surround the cranial cavity), while the double mutants for msx1 and msx2 are deficient in calvarial ossification, thus resembling the "Skull, general incomplete ossification" observed in GBH-exposed embryos by Dallegrove et al. Regulation of the Msx genes by retinoids is supported by (a) the identification of a retinoic acid-responsive element in the 50 flanking region of human MSX1 gene; and (b) functional in vivo evidence that indicates that endogenous retinoid signaling controls the spatial expression of this gene by inhibition. Therefore, it is conceivable that an increase in retinoid signaling upon exposure to GBH might inhibit msx expression, thus impairing the ossification of the cranial bones.

The other significant, dose-dependent effect of GBH exposure in rodent embryos described by Dallegrove et al. is "Caudal vertebrae: absent". It is well known to embryologists that exposure of mouse embryos to RA at a similar period of development produces agenesis of caudal vertebrae, which is caused by the down-regulation of posterior Hox genes.22

The arguments espoused by our critics do not and cannot rule out the possibility (which would be rather easy to check) that people exposed to GBH spraying accumulate glyphosate in their blood that can circulate and expose multiple tissues in the body to different concentrations of the chemical, producing different consequences. The vertebrate embryo is far from a black box that responds uniformly and monotonically to chemical insult. One possible example of the effects of spraying is the genotoxic effects reported in people exposed to agrochemicals and the effects in cultured cells exposed to dilutions of GBH that have been extensively studied by different laboratories in Argentina and Paraguay, Colombia, Ecuador, and France.23 25 These studies raise important questions regarding the safety of GBH and have never been adequately addressed.

It is an indisputable fact that our work to date shows a direct association between the abnormal expression of key molecular markers (shh, otx2, pax6, etc.) and alteredmorphogenesis caused by increased retinoic acid signaling. Most notably, effects of the alleged toxic doses of glyphosate that we have used in our experiments are rescued by the addition of a retinoic acid antagonist that blocks the activity of the retinoic acid receptor. This evidence that links GBH (and potentially other chemicals) to increased activity of the retinoic acid signaling pathway (a very well-known teratogen, even to industrial scientists) highlights the increased number of embryonic malformations and spontaneous abortions in populations subjected to spraying with GBH and other cocktails.

In addition, it should be noted that microinjection of pure glyphosate and incubation of embryos with dilutions of the GBH produced the same type of phenotypic changes. These can and must be addressed in the context of embryological strategies and the potential molecular mechanisms suggested by the effects. It is obvious that providing proof-of-principle is an important epistemological way to verify that ideas are plausible and feasible as a necessary precursor to future studies.

We contend that it is an epistemological error and a lack of scientific rigor to reject strategies and, more generally, to ignore alternative views about scientific evidence and developmental mechanisms that are key factors in assessing chemical safety for convenience, indolence, or profit. Speeches and position papers reassuring the public instead of scientific debate are not helpful. Worse yet is the propagation of misinformation. Sadly, such strategies are not new in the modern world and harken back to the debates about tobacco safety in the 1970s.

Andres E. Carrasco
Investigador Principal CONICET
Universidad de Buenos Aires

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NOTE ADDED IN PROOF
In the course of correcting the proofs for this reply, two new letters to the editor were submitted by Dr. Mulet (Universitat Politecnica de Valencia) and Dr. Palma (Asociacion Argentina de Productores en Siembra Directa, AAPRESID). The response to representatives of Monsanto, The Dow Chemical Company, United Phosphorus Inc., Nufarm Americas Inc., and Syngenta Ltd. is clear enough to avoid infinitely extending this exchange of letters that transcends the purely scientific interest. Despite this, it seems appropriate to make some comments:
(1) The present note is added in order to avoid falling into the habit of repetition.
(2) It seems pertinent to remark that one of the new letters was sent by a representative of the business association APRESSID, pretending to assume the role that would be expected by a peer review panel in the evaluation process of publication. This is achieved through disqualification in order to defend positions that have nothing to do with independent scientific activity. The doubts and skepticism expressed in the letters about dilution factors of the injected material, effect of pH, injection of substances in embryos, its meaning, its assessment, and interpretation are issues in Molecular Embryology and Developmental Biology that have been largely tested and accepted in these disciplines. Space and time excuse me for not being involved in discussions with interlocutors who are not familiar with this experimental discipline. These critics, besides impoverishing independent scientific discussion, show a light and inconsistent reading of our article.
(3) Finally, this is not the place for discussing epistemological issues about the value of different sources of knowledge.