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Abstract
Background The relationship between the use of biocides and insect repellents and the risk of hypospadias was examined in a large case–control study in the South East of England.
Methods A case–control study was carried out among 471 cases of hypospadias referred to surgeons, and 490 randomly selected population-based controls, born between 1 January 1997 and 30 September 1998. Telephone interviews were conducted between September 2000 and March 2003. The questionnaire included information on demographic, lifestyle and environmental factors, including the use of biocides and insect repellents, during pregnancy. A total biocide score was created from summing positive responses to an eight-item biocide exposure questionnaire.
Results The use of insect repellent (adjusted OR 1.81, 95% CI 1.06 to 3.11) during the first trimester of pregnancy was associated with risk of hypospadias, but none of the biocides, or indicators for them, except for the total biocide score for the highest two exposure categories (score 3: adjusted OR 1.73, 95% CI 1.02 to 2.94; and scores 4 and 5 combined: adjusted OR 2.98, 95% CI 1.01 to 8.78) showed statistically significant associations.
Conclusion The authors found an association between the use of insect repellent and total biocide score and risk of hypospadias. In particular, the use of insect repellent warrants further investigation, specifically in relation to type, content and frequency of use since this information was missing in the current study.
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Hypospadias is one of the most common congenital anomalies. Various risk factors have been associated with hypospadias including low birth weight,1–8 previous stillbirth,9 maternal age,10–14 educational level,5 subfertility,15 gestational age,2 4 16 smoking5 7 17 folate supplementation, occupational phthalate exposure and hair spray use.18
In addition, a number of studies have reported associations between pesticide exposure,16 19–21 specific pesticides such as diclofop-methyl (herbicide),16 o,p′-DDT, p,p′-DDT, lindane and mirex,9 22 and risk of hypospadias. Work or activities involving exposure to pesticides,6 17 23 being the wife of a farmer,24 engagement in agriculture9 or living in a rural area24 have also been associated with an increased risk of hypospadias. A temporal association between season of conception and risk of hypospadias corresponded approximately to the period at risk being the pesticides application period.24–26 No studies have investigated hypospadias and the use of insect repellents, which can contain, for example, N,N-diethyl-m-toluamide (DEET).27 28
The aim of the present study was to examine relationships between the use of biocides, or indicators of their use, and the use of insect repellents and the risk of hypospadias in a large case–control study in the South East of England.
Methods
The methods have been described previously.18 Briefly, we identified hypospadias cases born within a 21-month period (1 January 1997 to 30 September 1998) referred to surgeons in the South East of England. Cases were eligible if there was an abnormally positioned urethral orifice requiring surgery, with no major accompanying anomaly suggesting it was part of a syndrome. The study region included the health regions of North Thames, South Thames and the Anglian section of Anglia and Oxford, comprising 120 London boroughs and local authority districts. Surgical centres within the study region were visited as well as major surgical centres outside the area but within 50 miles, to identify cases born in the study region for whom treatment was sought beyond its boundaries. Forty of 41 surgeons operating on hypospadias in the study region participated; 731 cases were identified from surgeons' records and case notes. After initial contact with the mothers by letter, up to two further invitations were sent. Of the 731 mothers, 610 replied, of whom 471 (77%; 64% of total eligible cases) agreed to participate.
Controls born in the study region during the same period as the cases were randomly selected from the birth registry by the Office for National Statistics (ONS; London, UK). Ethics approval was given by the West Midlands Multi-centre Research Ethics Committee, Birmingham, UK. Following guidelines from the ethics committee, ONS asked the health authorities (now primary care trusts) to contact the general practitioners (GPs) of the control children. The GPs were then asked to pass on the invitation to the mother of the control child; in turn, the mothers were asked to contact the study team. In total, 1568 control mothers were selected, but letters were not sent out to 81 mothers. Of the possible 1487 invitees, 758 replied, of whom 490 (65%; 33% of total eligible controls) agreed to take part. We attempted to contact a sample (n=200) of the 729 mothers who had not replied and found that 144 (72%) had not received an invitation, mainly because the GP had not passed on the information (73%), the family had moved without a forwarding address (9.5%) or for other reasons.
Case and control mothers were interviewed by telephone between September 2000 and March 2003 using a standard set of questions, and answers were directly entered into a computer. The questionnaire included information on parental age, ethnicity, education, household income, family history of disease, pregnancy history, maternal occupation, use of biocides (including pesticides and insect repellents), pets, vegetarianism, folate supplements, smoking, and alcohol use during the first trimester of pregnancy. The study subject characteristics have been described elsewhere.18 There was no evidence of selection bias resulting from differential participation by socio-economic status represented by the Carstairs deprivation score.29 30
Statistical analysis
Exposure prevalence for cases and controls and unadjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Multiple logistic regression was carried out using Stata v 8.2. Exposure variables were included in the multiple regression models if they were associated with hypospadias at p<0.10 in the univariate model, and improved the fit in the multiple regression models. Highly correlated variables were not included together in the models to avoid multicolinearity. p Values are uncorrected for multiple comparisons. We created a total biocide score (range 0–8) by summing positive responses to the questions on living near an agricultural field (<1 mile) (1/0), use of garden pesticides (1/0), use of fly strips or sprays (1/0), use of ant powder (1/0), use of rat poison (1/0), spraying plants with insect spray (1/0), flea treatment of pets (1/0) and use of nit shampoo (1/0).
Results
In unadjusted analyses, biocide use and indicators for possible biocide exposure were not significantly associated with risk of hypospadias (table 1), except for the highest total biocide score (score 4 and 5 combined) (OR 4.05, 95% CI 1.45 to 11.29). The use of insect repellent (OR 1.76, 95% CI 1.08 to 2.86) showed a statistically significant association with the risk of hypospadias.
Unadjusted odds ratios for biocide use, potential biocide exposure, use of insect repellent and risk factors for hypospadias
After adjustment, the odds ratio for use of insect repellent increased slightly and remained statistically significant (OR 1.81, 95% CI 1.06 to 3.11) (table 2), as did odds ratios for the two highest exposure categories of the total biocide score (3, and 4 and 5 combined), respectively OR 1.73 (95% CI 1.02 to 2.94) and OR 2.98 (95% CI 1.01 to 8.78) (p value for trend=0.058). The use of naphthalene and the use of ant powder were associated at p<0.10 in the unadjusted analyses, and were carried forward to the adjusted analyses; odds ratios were 2.04 (95% CI 0.91 to 4.56) and 1.33 (95% CI 0.92 to 1.93), respectively (table 2).
Adjusted odds ratios for use of insect repellent, certain biocides and risk of hypospadias in four separate multivariate models
Discussion
Our finding of an increased risk of hypospadias associated with exposure to insect repellents is novel. Insect repellents can contain, for example, N,N-diethyl-m-toluamide (DEET)27 or permethrin.28 High doses of DEET are toxic and it can cross the placental barrier,31 but its consequences are not well understood.32 33 Reviews by Osimitz and Murphy,34 Goodyer and Behrens35 and Koren et al36 concluded that there were no or low health risks from exposure to insect repellents, including adverse pregnancy outcomes, while McGready et al31 found no adverse neurological, gastrointestinal or dermatological effects in almost 900 pregnancies, and no adverse effects on survival, growth or development at birth or at 1 year in 50 pregnancies where the mother was using DEET. A combination of DEET, permethrin and pyridostigmine bromide (eg, used as a prophylactic treatment against nerve agents) caused testicular germ-cell apoptosis (cell death) in rats37 and may have consequences for reproduction. Application to the skin of DEET has been shown to cause problems of development or even death of the fetus/embryo of female rats.27 A reduced fetal birth weight has been observed in litters of rats exposed to high doses of DEET during pregnancy.38 Embryo development was also compromised by permethrin when associated with other insecticides during early mouse pregnancy and at very low doses.39
Concerning biocide use, some6 9 16 19 20–26 but not all11 17 40–42 previous studies have reported an association between direct or indirect measures of pesticide exposure and hypospadias. In the present study, we found no association with use of biocides or indicators for its use except for total biocide score, but it is possible that total biocide score is acting as a proxy for exposure to other agents with a putative effect on hypospadias risk, potentially confounding our results, as pesticide users tend to be less risk averse than non-pesticide users, which may lead to greater use of other chemicals.43
Our study has limitations. We relied on questionnaire data rather than measurement for exposure assessment. We did not have information on the type of insect repellent used nor its content, and the lack of measurements of biocides in the environment or in biological samples may have led to exposure misclassification and, if non-systematic, consequent reductions in power to detect possible associations; we did not ask for specific names of biocides or active ingredients, or visit the homes, which may also have led to misclassification, further limiting interpretation.43–45 Recall bias may have occurred, but it is difficult to estimate to what extent, although no previous study has linked hypospadias with insect repellents, and therefore if there is recall bias, it may affect the pesticide estimates more than insect repellents. Furthermore, the analyses were not adjusted for multiple testing, so it is possible that our positive findings for insect repellents and total biocide score reflect chance associations.
In summary, we found a significant association for risk of hypospadias with the use of insect repellents and total biocide score, but not with the use of individual biocides or indicators for its use. Further work should be conducted on the possible reproductive effects of insect repellents, with consideration of the type, content and mechanisms of action of specific formulations, and the current findings need to be replicated before firm conclusions can be drawn.
What this paper adds
Relatively little is know about the environmental risk factors for hypospadias.
This study shows for the first time evidence of an association between insect repellent use and hypospadias.
Further, it also shows an association between multiple pesticides use and hypospadias.
Resources are needed to further investigate these risk factors, particularly insect repellent use.
No changes in practices are recommended until further evidence is available.
Acknowledgments
We thank Marie-Louise Dudley and Claire Brown for conducting the interviews, James Stewart-Evans for database cleaning, the Office for National Statistics for providing the names and contact details of controls, and the surgeons, GPs and mothers who participated in the study. We are grateful to Professor Ieuan Hughes, Mr Pierre Mouriquand MD and Professor Duncan Wilcox MD for invaluable clinical input.
References
Footnotes
Funding The study was funded by a grant from the UK Health and Safety Executive/Department of Health/Department of the Environment, Transport and The Regions/European Chemical Industry Council (CEFIC). Paul Nelson was supported by a Wellcome Trust Research Training Fellowship in Clinical Epidemiology and also in part by the North Thames Training Programme in Public Health Medicine. The study sponsors had no role in the study design, data collection, analysis and interpretation, or writing of the paper.
Competing interests None.
Ethics approval Ethics approval was given by the West Midlands Multi-centre Research Ethics Committee, Birmingham, United Kingdom.
Provenance and peer review Not commissioned; externally peer reviewed.