Dan Dietrich

Human and Environmental Toxicology University of Konstanz

CAAT-IPAM-ISS Symposium Rome 17 December 2012

www.irish-health.com 2001

2/8/2013 Dietrich Environm. Toxicology 3

„Are oestrogens involved in falling sperm counts and

disorders of the male reproductive tract?“

(Sharpe and Skakkebaek, Lancet, 341:1392-5, 1993)

„Feminized responses in fish to environmental estrogens“

(Sumpter, Toxicol. Lett, 82-83:737-42, 1995)

„Developmental effects of endocrine-disrupting chemicals in

wildlife and humans“

(Colborn, v. Saal, Soto, Env. Health Persp. 101:378-84, 1993)

„Sex-change chemicals in Potomac“, BBC,

Jan.18, 2007

“This [BPA panel] was the most dysfunctional thing I’ve ever sat on,” says biologist Theo

Colborn ........“although the science is stronger, the tests EPA is requesting are inadequate. “A

chemical like BPA could easily be missed in the assays they have selected,” she says (Nature,

April 2010)

Toxic science

„The gap between the methods used in industry and those of

basic researchers can be traced back to 1993, when vom Saal

and his colleagues showed how organisms can be exquisitely

sensitive to tiny amounts of hormone- like chemicals during

development“.......... (Nature, April 2010)

„Why public health agencies cannot depend on good laboratory practices

as a criterion for selecting data: the case of bisphenol A“

Myers et al Environ Health Perspect, 117:309-15, 2009

„Bisphenol-A Exposure during Pregnancy Disrupts

Glucose Homeostasis in Mothers and Adult Male

(Mouse) Offspring“, Alonso-Magdalena et al, Environ

Health Perspect, 2010 September; 118(9): 1243–

1250„Perinatal exposure to bisphenol a alters early

adipogenesis in the rat“

Environ Health Persp 117:1549-55, 2009

Statistical „association“: any relationship between two measured

quantities that renders them statistically dependent i.e. they are

dependent on one another with some degree of likelihood,

however, an association does not connote that “cause and

effect” exist

EAS associated with „endocrine disruption“ i.e. increased

incidences of specific adverse health effects in humans: BPA,

phthalates, PCBs, Dioxins and DDT and metabolites

........Much of that fear was fueled by a 1992 Danish study that seemed to show that sperm counts had

declined by 50% globally from 1938 to 1991, and that the trend would continue into the future........ The study was criticized by many researchers for its flaws — including the length of time men abstained from ejaculation before their semen was collected, which can affect sperm quality — but it was also extremely influential, cited by more than 1,000 subsequent studies...... The same Danish group that published the original study has been collecting and analyzing annual semen samples from 18-year-old Danish men who were being examined for their military fitness. Over the past 15 years, 5,000 men have provided semen for analysis — a much better data pool than was involved in previousresearch................

Epidemiology • Volume 22, Number 5, September 2011The data, which were published in the American journal Epidemiology, show virtually no evidence of declining spermquality, with levels barely changing from 1996 to 2010........

“If at the outset of the experiment we strongly believe that storks bring babies in spring (thus completely ignoring all facts provided by reproductive biology and common sense) and we determine stork number and frequency in a given area in Europe with the number of babies delivered, we can state that due to the highly significant correlation of the two observations, the causal relationship between storks and baby deliveries is proven. Indeed, in Kosovo, Albania and other areas of the Balkans the number of storks and babies per year is highest and thus also demonstrates an irrefutable robust statistically significant correlation, whereas in central Europe the number of storks as well as the number of births have been at a minimum over the past decades.”

� Epidemiological studies versus experimental science

� Epidemiological studies fraught with assumptions and inappropriate assertions and designs, e.g. the French study 2012:

„Socioeconomic status could not be controlled for. Despite universal access to medical servicesin France, couples undergoing ART are expected to have a higher educational level onaverage compared with those of the general population. Therefore, the real values in thegeneral population could be slightly lower than those presented and the decrease possiblystronger, as the population study is less likely to smoke or be overweight, two factorsknown to impair semen quality“.

� Merchands of doom mix data from animal and humanstudies as fit for their purpose and then design their ownexperiments accordingly, e.g. the French study 2012:

„...growing biological, toxicological, experimental and human exposure data support the endocrine disruptors’ hypothesis assuming that fetal exposure to endocrine disruptors could impair reproductive outcomes.....“

� Primarily argumentation from animal experiments!

� Use of studies with domination of effects, i.e. descriptive toxicology (e.g. OECD TG 407) and only very few limited mechanistic and evidence based approaches. While in reality a selection of well designed mechanistic studies with maximum insight as to the relevance of the data for the human are needed.

� Biased reporting or over-interpretation of study results (Sena et al., 2010) e.g. insufficient consideration of species differences, dose-response principles, experimental design issues, etc., often in conjunction with highly visible and thus politically “in” research areas (e.g. obesity research).

� A penchant to holding onto the “false-safety” of traditional i.e. historically approaches and thus repeating past mistakes rather than embracing modern science – “the Flat-Earth Society of Toxicology”

� Inadequate and counter “common sense” use of

weight-of-evidence in the interpretation and

reporting of risk to humans: “fish, rats and mice

equal humans in the reproductive physiology, thus

also toxicokinetics and dynamics?”

� Individuals and groups in society that are willing to

interpret and extrapolate toxicological data that

causes exaggerated concern

� Experts with extremely disparate expertise involved

in the risk assessment process

� The example of DES elegantly demonstrates that even for themost potent EAS in humans (with regard to endocrinedisruption) known to date, it is primarily a “high dose, specificactivity, and prolonged time during a critical period principle”that governs the manifestation of “endocrine disruptiveeffects” in humans.

� The current data IN HUMANS for steroids employed ascontraceptives, SERMs, glitazones (4mg/d) or ketoconazole(600-1000mg/d) DO NOT provide any evidence that even inshort-term overdose situations during gestation, endocrinedisruptive effects occur in the offspring

� All current toxicological information to date strongly suggestthat the routine rodent models are inadequate for detection ofEAS and assumed endocrine effects in humans

� Introduced in the 1920‘s. commercial since 1930‘s

� Epidemiologic cross-sectional studies:

� Negative correlation betwen phthalate metabolites in mothers urine and male babies anogenital distance, penile volume/length and cryptorchidism (Swan 2008, Bustamante-Montes et al 2008)

� Complete opposite findings (Huang et al, 2009)

� Problem with all studies:

� limited control or adequate incorporation of confounding factors

� Absence of any suggested mechanism that could explain the findings

� In vitro human ERα and shbg binding:

� No binding of DBP (Freyberger et al 2010, Hodgert-Jury et

al 2000)

� In vitro human ERα and AR studies (agonistic and

antagonistic mode):

� No activity of DBP (Witters et al 2010; Freyberger et al

2010)

� Human in vitro studies with di(n-butyl)phthalate (DBP) /monobutylphthalate (MBP) (metabolite) on Leydig cell steroidogenesis (Hallmark et al 2007):� Use of human fetal testis explants

� No adverse effects observed

� Human fetal testis xenografts are resistant to phthalate-induced endocrine disruption (Heger et al. 2010)

� In vivo studies with rats exposed in utero, and twin male marmosets to DBP 4 days post-partum:� Inhibition of steroidogenesis (lowered testosterone levels)

� Rat in vitro studies with fetal testis explants:� Inhibition of steroidogenesis (Heger et al., 2012)

� Conclusion: NO RISK of DBP for humans, but bad for rats, mice and monkeys

� Large cohort study associated BPA exposure with higher risk

for diabetes and heart disease (Lang et al 2008; Melzer et al

2010) – interpretation derived from findings in mice

(Alonso-Magdalena et al 2010) and rats (Somm et al 2009) –

no credible mechanism provided.

� Cross-sectional study found no association between BPA

exposure and reproductive parameters in fertile men

(Mendiola et al 2010) – alluding to known potential

endocrine activity in vitro and controversially discussed in

vivo results from rodent studies.

� In vitro competitive binding to human ERα and ERβ : 7,3 x 10-4 M and 7,5 x 10-3 M (Chapin et al, 2008)

� In vitro agonism in transactivation assays:

� MVLN cells with constitutive ERα - no activity (Freyberger & Schmuck, 2005)

� MCF7 cells transiently transfected with ERα & ERβ – 7,1 x 10-7 M and 4,5 x 10-7 M (Nicolas 2003; Matthews et al 2000)

� In vitro binding to SHBG

� Nearly absent – at detection level of assay.

� Conclusions: in vitro assays with “human systems” suggest that it would take a 10 000 –fold higher concentration of BPA than E2 in situ in order to displace 50% of E2 binding at the ER.

� Despite a high dietary load of BPA in volunteers, in 83% of the volunteers BPA was below the LOD (1.3 x 10-9 M) in serum (Teeguarden et al 2011)...... „the concentrations in the blood that we calculate are 1,000 to 1 million times lower than the levels that would be required to bind the various estrogen receptors that are believed to cause BPA toxicity and endocrine disruption.”

� “In a nutshell, we (Teeguarden et al) can now say for the adult human population exposed to even very high dietary levels, blood concentrations of the bioactive form of BPA through the day are below our ability to detect them, and orders of magnitude lower than those causing effects in rodents exposed to BPA.”

� „A PBPK-based reverse dosimetry approach was employed to calculate upper bound concentrations of the bioactive form of BPA in the blood of human volunteers exposed to high dietary conc. BPA. The BPA levels detected were orders of magnitude below levels required for activation of estrogen receptors.” Shankaran and Teeguarden 2012.

� “Receptor binding is a plausible mechanistic step in humans only if serum concentrations of the bioactive (unconjugated) form of BPA are near the disassociation constants for estrogen receptors. Human exposure to BPA is widespread, but efficient first pass glucuronidation limits the bioactive fraction of BPA to ~0.1 % of total BPA. Three independent methods used to calculate the plausible human serum concentrations of bioactive, unconjugated BPA show that these concentrations are multiple orders of magnitude below existing analytical detection limits, indicating that unconjugated BPA should rarely be observed in the serum of adult humans or children at current exposure levels.“Teeguarden 2012.

� „The mean or median concentrations of bioactive BPA are multiple orders of magnitude below the disassociation constants for all estrogen receptors considered candidates for mediating the toxicity of BPA.” Teeguarden 2012.

� „Infant exposure scenarios calculated (Breast-milk, BPA-free, and polycarbonate), demonstrated that exposures of infants did not exceed the toxicity criteria values, and in some cases they were orders of magnitude lower. Although exposures calculated in the breast milk category were the lowest overall, all scenarios resulted in exposures that were below the toxicity criteria guideline values. Overall, the evidence does not support a reduction of risks from using BPA-free products.“ Peterson and Goodman, 2012

� In April 2009: The French Minister for Health (Roselyne Bachelot) recently stated that the Canadian decision to ban Bisphenol A (BPA) was only due to public pressure, and was not based on ‘serious science’..

� In November 2011: The French Minister of Health (Xavier Bertrand) offers support to a law that would ban the use of Bisphenol A in all food packaging by 2014

EFSA 2011: In a response to the French ban, states that there is no new evidence that would demand a ban.EFSA April 2012: EFSA launches full re-evaluation of BPA focussing on exposure and possible „low dose effects“.

� In vivo and in vitro experiments with animals and animal derived cells provide potential risk information on the risk of compounds (DEP; BPA) for the animals but not for humans.

� In vivo and in vitro experiments with animals and animal derived cells provide data that is misused for „political & polemic“ purposes to exagerate and describe inexistent risk for humans.

� Appropriate use ot toxicological knowledge from in vitro experiments with human cells and organs systems coupled with proper toxicokinetic modelling in humans will provide sufficiently robust data to allow for a highly reliable risk description and calculation.

Courtesy: Gary Larsson