1. Pesticides, lead and solvents: pregnancy outcome and fertility Markku Sallmén Finnish Institute of Occupational Health

2. Pesticides n The most extensively used group of toxic chemicals n ca. 600 different active ingradients n about 50,000 various formulations n simultaneous exposure to different chemicals typical n pesticide formulations may contain solvents

3. Pesticide exposure in farming n Most of the exposure occurs via the skin n Some pesticides degrade on the leaves into more toxic compounds with even stronger penetration capacity n Inhalation during applications n Mixing, formulation, spraying, handling of treated plants, cleaning of equipment and clothes, storage

4. Pesticides Comparison between studies difficult n Different pesticides are used for different purposes and in different areas n Exposure levels vary considerably; the highest exposures occur in developing countries in poorly controlled circumstances n What is the affected gender?

5. Pesticides shown adverse effects on spermatogenesis n DBCP n 2,4-D (2,4-dichlorophenoxy acetic acid) n ethylene dibromide n chlordecone n carbaryl

6. Pesticides and semen quality StudyOutcome n Larsen et al 1998 (longitudinal study)–multiple outcomes n Larsen et al 1999 (organic/traditional)–multiple outcomes n Padungtod et al 1999 (factory workers)+aneuploidy n Tielemans et al 1999 (inf clinic clients)(+)combined quality n Juhler et al 1999 (dietary pesticides)–dead spermat. n Oliva et al 2001 (infertility clinic clients)+multiple outcomes n Abell et al 2000 (greenhouse workers)+concent./morphol n Wong et al 2003 (population based)+oligozoospermia n Dalvie et al 2003 (DDT, malaria control)– multiple outcomes n Swan et al 2003 (population based)+summary of many n Sanchez-Pena et al 2004 (organophos.)+ sperm chromatin + = reduced semen quality, (+) suggestive association – = no association

7. Pesticides and male fecundability n StudyAssociation n de Cock et al 1994+ n Larsen et al 1998- n Thonneau et al 1999- n Curtis et al 1999(+), gender? n Petrelli et al 2001+ n Sallmén et al 2003(+) + = reduced fecundability, (+) suggestive association

8. Male pesticide exposure and couples undergoing IVF treatment (Tielemans et al 1999, 2000) n Reduced fertilization capacity –OR for confirmed exposure 0.38 (0.19-0.78) –OR for potential exposure 0.54 (0.29-0.99) n Improved implantation rate –OR for high/moderate exposure 3.31 (1.25-8.80) –OR for high(strict) exposure 1.57 (0.33-7.44) n Summary effect?

9. Male pesticide exposure and spontaneous abortion Savitz et al 1994, reviewed 14 studies: n elevated RR in >1 study: YES n RR > 1.5YES n Evidence from high quality studies:NO Thereafter mainly US studies have shown associations with spontaneous abortion

10. Male pesticide exposure and congenital malformations García 1998, a review on occupational exposure and congenital malformations: n 17 studies n 4 studies showed an association Conclusion: Inadequate evidence for either establishing a relationship between pesticides exposure in humans and birth defects or for rejecting it.

11. Male pesticide exposure and congenital malformations Pesticide applicators in the Red River Valley of Minnesota (Garry et al, 1996, 2002) n 1996 a register-based study - excess in birth defects - seasonal pattern n 2002 a cross-sectional interview study - rate of birth defects 7.6% (spring) vs. 3.7% other season - herbicides: risk of birth defects - fungisides: determination of sex of the child Salazar-Garcia et al 2004 (DDT); OR 3.4 (1.2-9.5)

12. Conclusion: Male pesticide exposure n High exposure associated with reduced semen quality n Inconsistent findings on fecundability n Small number of TTP studies n The findings of the new studies seem to have added the evidence that male (or parental) exposure to pesticides is associated with adverse pregnancy outcome

13. Pesticides and female fertility n StudyAssociation n Fuortes et al 1997+ n Greenlee et al 2003+ n Curtis et al 1999+ (6 of 13 pesticides) n Abell et al 2000+ + = reduced fertility

14. Summary:Female pesticide exposure and fecundability n There is evidence that female high exposure to pesticides is associated with reduced fecundability n This conclusion is based on small number of studies, however

15. Female pesticide exposure and adverse pregnancy outcome Nurminen 1995, and Garcia 1998 reviews: n Conclusion: the epidemiologic evidence is inconclusive as regards the risk of adverse pregnancy outcome n Conclusion: Inadequate evidence for either establishing a relationship between pesticides exposure in humans and birth defects or for rejecting it.

16. Female exposure to specific pesticides and spontaneous abortion Exposure/StudyAssociation (OR) Hexachlorobenzene (serum, ng/ml): Jarrell et al 1998<11.6p=0.113 >14.1p=0.02 DDT; serum p,p'-DDE measured: Korrick et al 2001 (each ng/g)1.131.02-1.26 Longnecker et al 2003 (per 60 µg/L) 1.41.1-1.6

17. Pesticides and congenital malformations or foetal death n Female: Exposure assessment n Pastore et al 1997 (popul based case-control)+female occup exp n Bell et al 2001 (popul based case-control)+residence/application n Longnecker et al 2002 (popul based case-c)(+)p,p'-DDE n Ribas-Fito et al 2003 (birth cohort, neurodev.)+p,p'-DDE n Medina-Carrilo et al 2002 (popul based case-c)+female occup exp n Bell et al 2001 (fetal death, popul case-cohort)(+)residence/application n Male: n Garcia et al 1998 (case-referent)+interview, experts n Garry et al 2002 (cross-sectional, popul based)+spring vs. other n Regidor et al 2004 (population based)+season n Either gender: n Kristensen et al 1997(cohort )+agricultural census n Schreinemachers 2003 (population b.)+ecologic + = increased risk, (+) suggestive association

18. Pesticides conclusion: females n Several studies with varying outcomes, pesticides, level of exposure, affected gender, and quality of the study n High exposure seems to be associated with increased risk n Exposure should be restricted through efficient protection n Should pregnant be transferred?

19. Lead: Pregnancy outcome and fertility

20. Simultaneous exposure to several metals n Studies around Rönnskär copper smelter ð excess of spontaneous abortion, and stillbirths in pregnancies of wives of men exposed to lead, copper, zinc, gold, silver, cadmium, mercury, arsenic, and sulfur dioxide ð carry-home exposure to the wives remains a possible alternative explanation

21. Population-based studies n Rachootin and Olsen 1983 ð case couples examined or treated for a problem of infertility at Odense University Hospital ð questionnaire information on job and exposure ð female exposure to lead, mercury, and cadmium were associated with infertility

22. Maternal lead exposure and spontaneous abortion B-Pb µmol/LOR95% CI A retrospective study among biologically monitored workers:  Taskinen 19880.5-3.10.80.5-1.4 Measured within a year of pregnancy: >1.41.90.4-9.4 A prospective study (B-Pb measured at gestational age 4-12 w):  Borja-Aburto0.24-0.482.3 -- et al. 19990.49-0.725.4 -- > 0.7212.2 --

23. Maternal lead exposure and fecundability n Sallmén et al 1995 (study among women biologically monitored for exposure to lead) n B lood leadFDR95% CI not exposed1.00reference <0.5 µmol/l0.930.56-1.57 0.5-0.9 µmol/l0.840.45-1.45 1.0-2.4 µmol/l0.800.42-1.54 Eight most heavily exposed subjects: 1.4-2.4 µmol/l0.530.19-1.52

24. Maternal lead exposure and cognitive development; prospective studies StudyMean Blood LeadEffect Boston7.37 µg/dl+ Cincinnati4.6 --->14.1+ Cleveland5.8 ---> 6.5- Port Pirie14.4->21.2->17.6+ Sydney9.1->8.1->12.5- Yugoslavia14.4--->24.3(+/-) 1 µmol/L = 20.7 µg/dL

25. Lead and semen quality Apostoli et al 1997 (a review): n Exposure to lead at blood lead 1.9 µmol/L (40 µg/dL) is hazardous for male reproductive function n reduced sperm count, volume, and density n changed sperm motility and morphology n a modest effect on endocrine profile is possible Viskum et al 1999: n The effect is, at least partially, reversible

26. Studies on birth rates and male exposure to lead StudyPbB level Effect n Selevan et al. 19841.2 µmol/l+ n Coste et al. 19911.9 µmol/l- n Gennart et al. 19922.2 µmol/l+ n Lin et al. 1996 – duration of exp. >5 years,2.4 µmol/l+ n Bonde and Kolstad 19971.8 µmol/l- + reduced fertility, - no effect

27. Relative risk (RR) of infertility and male exposure to lead; Sallmén et al 2000 Estimated PbB RR 95% C.I. µmol/L 0.5-0.9 1.27 1.08-1.51 1.0-1.4 1.35 1.12-1.63 1.5-1.91.37 1.08-1.72 2.0-2.41.50 1.08-2.02 > 2.51.90 1.30-2.59

29. Male lead exposure and spontaneous abortion Study PbB µmol/L OR 95% CI Selevan1984 1.2 - 1.91.50.8-3.0 2.0 - 2.91.10.6-1.9 > 3.00.90.3-2.3 Al-Hakkak19861.8 - 4.63.0p<.01 Lindbohm19911.0 - 1.41.00.6-1.7 1.5 - 1.81.30.5-3.4 > 1.91.60.6-4.0 Alexander19961.2 - 1.81.00.6-1.7 > 1.90.70.4-1.5

30. Male lead exposure and congenital malformations or perinatal death StudyPbB µmol/L OR 95% CI M: Sallmén 1992> 1.02.40.9-6.5 P: Kristensen 1993exposed2.41.2-4.9 P/M: Alexander 19961.2 - 1.82.90.6-13 > 1.92.50.5-11 M=malformation study P=perinatal death study

31. Solvents: pregnancy outcome and fertility From a presentation of: Dr. Marja-Liisa Lindbohm Finnish Institute of Occupational Health

32. Organic solvents important occupational reproductive hazards: widely used in various fields of industry volatile liquids absorbed via inhalation and through the skin most solvents traverse the placenta diverse group of compounds

33. Reproductive effects of solvents in several human studies exposure related to reduced fertility spontaneous abortions birth defects low birth weight in some studies exposure related also to menstrual disorders and change in hormone levels pregnancy-induced hypertension neurobehavioral performance reduced semen quality childhood cancer

34. Maternal exposure to solvents in some occupations and spontaneous abortion

35. Cumulative percentage of pregnancies by maternal exposure to solvents (Sallmén et al 1995)

36. Industries and individual solvents related to reduced fertility or adverse pregnancy outcome Dry cleaning: tetrachloroethylene Semiconductor industry: ethylene glycol ethers Shoe and leather industry: aliphatic hydrocarbons, toluene, hexane Laboratory work: toluene, xylene, chloroform

37. Ethylene glycol ethers used in paints, dyes, lacquers, waxes etc. Methoxyethanol embryotoxic and teratogenic in mice and rats teratogenic effects seen at doses that do not cause overt maternal toxicity Ethoxyethanol induced skeletal defects in rats, multiple defects in rabbits (inhalation exposure) in rats, impaired performance in behavioral tests

38. Spontaneous abortion, fertility and maternal exposure to mixtures containing ethylene glycol ethers in semiconductor industry (Correa et al. 1996, Chen et al 2002) OutcomeExposureRR/FR95% CI Spontaneous abortion: low1.0 0.6 – 1.7 Correa et almedium1.4 0.8 – 2.6 high2.8 1.4 – 5.6 Time-to-pregnancy: Correa et almedium0.8 0.6 – 1.1 high0.7 0.4 – 1.1 Chen et alExposed0.59 0.37 – 0.94

39. Conclusions: Effects of solvent exposure on the reproductive health of women High exposure to solvents increases the risk of spontaneous abortion and decreases fertility The findings on birth defects less conclusive, but suggesting adverse effects Particular solvents associated with adverse effects: ethylene glycol ethers, tetrachloroethylene, toluene

40. Exposure assessment and recommendation Assessment of solvent exposure with industrial hygienic measurements or biological monitoring In some countries the guidelines recommend that solvent exposure should not exceed 10% of the threshold limit value during pregnancy Reproductive effects have been used as the basis for some TLVs

41. Exposure to solvents and semen quality or hormone levels Solvents: reduced sperm quality and decreased implantation rate Ethylene glycol ethers and 2-bromopropane: reduced sperm count Styrene and acetone: sperm anomalies Toluene and solvents in general: decrease of hormone levels

42. Solvent exposure and seminal characteristics in 225 men who had their first infertility consultation (Oliva et al 2001) Seminal charateristicOR95% CI Seminal volume >3.8 ml2.40.8 – 7.6 Sperm concentration <1x10 6 /ml2.70.9 – 8.3 Sperm output <3x10 6 2.50.8 – 7.9 Sperm motility <50%3.11 – 9.5 Sperm morphology <30%3.01.0 – 9.0

43. Cumulative percentage of pregnancies by paternal exposure to solvents (Sallmén et al. 1998)

44. Ethylene glycol ethers and male fertility

45. Paternal exposure to solvents and spontaneous abortion

46. Paternal solvent exposure and pregnancy outcome Inconsistent findings on the effects of exposure on low birth weight An excess of birth defects in the children of male painters, but not in the children of other exposed workers Some evidence for childhood leukemia or nervous system cancers, and paternal exposure to solvents Evidence inconclusive, although suggestive associations noted

47. Conclusions: Effects of solvent exposure on the reproductive health of men Solvent exposure related to reduced sperm quality Ethylene glycol ethers harmful for male reproductive system Carbon disulfide related to decreased libido and potency in men No clear association between solvent exposure and decreased fertility

48. Male pesticide exposure and spontaneous abortion Study Association (OR) Arbuckle et al 1999, Ontario Farm Family Study, phenoxy herbicides <20 weeks of gestation1.10.6-1.9 <12 weeks of gestation 2.51.0-6.4 husband not using protective 5.00.7-36.2 equipment Petrelli et al 2000 3.81.2-12.0 Crisostomo et al 2002 6.2 1.4-27.9 Salazar-Garcia et al 2004 (DDT)1.51.1-2.1

49. Male/female pesticide exposure and spontaneous abortion Arbuckle et al 2001 Ontario farm population Timing of exposure / affected gender: preconceptionalearly (<12 weeks) abortions (male exposure?) postconceptionallate (12-19 weeks) abortions (female exposure?)

50. Male pesticide exposure and spontaneous abortion Pesticide applicators in the Red River Valley of Minnesota (Garry et al, 2002) Fungicides: 1.6-2 -fold increase in risk for miscarriage/fetal loss Herbicides: increased risk in first-trimester miscarriage The overall toxicity data suggest a male- mediated event Also, women engaged in pesticide application were at risk

51. Female pesticide exposure and spontaneous abortion StudyAssociation Bell et al 2001pesticides showed no strong association with fetal death (ORs from 0.9 to 1.4) Exposed: lived in 1+ 8 adjacent sq miles from the application

52. Parental pesticide exposure and congenital malformations Kristensen et al 1997; a study among Norwegian farmers Exposure to pesticides associated with: spina bifida hydrocephaly limb reduction cryptorchidism hypospadias

53. Fecundability Density Ratio (FDR) of Pregnancies by Father’s Exposure to Lead; Sallmén et al 2000 Estimated PbB µmol/l N FDR 95% C.I. 0.5-0.9 203 0.92 0.73 - 1.16 1.0-1.4 79 0.89 0.66 - 1.20 1.5-1.8 21 0.58 0.33 - 0.96 > 1.9 23 0.83 0.50 - 1.32 > 1.5 (combined) 440.700.47 - 1.01

54. Reproductive effects of some solvents in animal tests ethoxyethanol: teratogenic and spermatotoxic effects methyl alcohol: teratogenic effects methyl ethyl ketone: decreased fetal body weight n-hexane: testicular lesions xylene and white spirit (prenatal exposure): learning and memory defects

55. Maternal exposure to solvents and pregnancy outcome: a meta-analysis (McMartin et al. 1998) 559 articles identified; around epid. studies 90 Spontaneous abortions five studies included (n=2899) summary OR=1.25 (95% CI 0.99 – 1.58) Birth defects five studies included (n=7036) summary OR=1.64 (95% CI 1.16 – 2.30) Reasons used for the exclusion of (several potentially important) studies criticized

56. Tetrachloroethylene used as a dry cleaning agent and degreaser passes across the placenta animal studies: no clear teratogenic effects signs of fetotoxicity observed in some studies a two-generation study found decrease in litter size and postnatal survival at 300 ppm propably carcinogenic to humans (2A, IARC)

57. Maternal exposure to tetrachloroethylene in dry cleaning and spontaneous abortion

58. Toluene used in paints, inks, coatings, adhesives, and in the leather, rubber and graphic industries low birth weight, microcephaly and facial abnormalities in children of women abusing toluene by inhalation during pregnancy reduced birth weight in prenatally exposed rat pups effects on cognitive function reported in rats after prenatal exposure

59. Maternal exposure to toluene and spontaneous abortion or reduced fertility PopulationRelative risk95% CI Spontaneous abortionsOR Pharmaceutical factory w.1.90.6 – 6.4 Laboratory workers4.71.4 – 15.9 Monitored workers1.40.4 – 4.9 *Audio speaker factory w.2.81.3 – 5.9 Reduced fertility (ttp)FDR Monitored workers0.710.40-1.26 *Printing industry workers0.470.29-0.77

60. Maternal exposure to solvents and oral clefts

61. Methodological issues in epidemiologic studies on solvents response rates satisfactory in most studies confounding usually, but not always controlled for outcome data mainly from the medical records data on exposure usually based on workers’ own reports - underreporting ? in some studies exposure assessed by experts exposure to mixture of solvents common small sample size in studies on birth defects and individual solvents

62. Reproductive endocrine effects in women with fuel/solvent exposure (Reutman et al 2002) Outcome: urinary endocrine markers related to nonconceptive menstrual cycles (N=63) Exposure assessment: levels of aliphatic and aromatic hydrocarbons (HCs) in exhaled breath Result: preovulatory luteinizing hormone level significantly lower among women who had higher internal doses of aliphatic HCs Conclusion: compounds in fuel and some solvents may act as reproductive endocrine disruptors

63. Genetic susceptibility to benzene and shortened gestation (Wang et al 2000) Aim: examine whether the association between exposure and outcome is modified by two susceptibility genes CYP1A1 and GSTT1 responsible for detoxification of solvents (542 mothers) Results: benzene exposure associated with a decrease in mean gestational age when stratified by the maternal CYP1A1 genotype, the decrease was significantly greater for the AA group than for the AA/aa group Provide evidence of gene-environment interaction

64. Carbon disulfide exposure and the prevalence of sexual complaints

65. Solvent exposure and count of motile sperm (Cherry et al 2001) A case-referent study of 656 infertility patients Aim: to examine whether cases with low motile sperm count (<12x10 6 ml) were more likely than referents to have had exposure to solvents Exposure assessment: job exposure matrix of previous studies on solvent exposure Results: OR 2.1 (95% CI 1.2-3.4) for moderate exposure OR 3.8 (95% CI 1.4-10.6) for high exposure