Association Between Thimerosal-Containing Vaccine and Autism

BRIEF REPORT

Anders Hviid, MSc Context Mercuric compounds are nephrotoxic and neurotoxic at high doses. Thi- Michael Stellfeld, MD merosal, a preservative used widely in vaccine formulations, contains ethylmercury. Thus it has been suggested that childhood vaccination with thimerosal-containing vac- Jan Wohlfahrt, MSc cine could be causally related to neurodevelopmental disorders such as autism. Mads Melbye, MD, PhD Objective To determine whether vaccination with a thimerosal-containing vaccine is associated with development of autism. IGH DOSES OF MERCURIC COM- Design, Setting, and Participants Population-based cohort study of all children pounds are nephrotoxic and born in Denmark from January 1, 1990, until December 31, 1996 (N=467450) com- neurotoxic.1 Thimerosal, an paring children vaccinated with a thimerosal-containing vaccine with children vacci- organic compound that con- nated with a thimerosal-free formulation of the same vaccine. tainsH ethylmercury, has been widely used Main Outcome Measures Rate ratio (RR) for autism and other autistic-spectrum since the 1930s as a preservative in cer- disorders, including trend with dose of ethylmercury. tain vaccines. In the 1990s, an increas- Results During 2986654 person-years, we identified 440 autism cases and 787 cases ing number of different vaccines con- of other autistic-spectrum disorders. The risk of autism and other autistic-spectrum taining thimerosal were introduced in disorders did not differ significantly between children vaccinated with thimerosal- immunization schedules around the containing vaccine and children vaccinated with thimerosal-free vaccine (RR, 0.85 [95% world, and thus the average cumula- confidence interval {CI}, 0.60-1.20] for autism; RR, 1.12 [95% CI, 0.88-1.43] for other tive exposure to thimerosal in infants has autistic-spectrum disorders). Furthermore, we found no evidence of a dose-response association (increase in RR per 25 µg of ethylmercury, 0.98 [95% CI, 0.90-1.06] for increased in recent years. This has led autism and 1.03 [95% CI, 0.98-1.09] for other autistic-spectrum disorders). to the suggestion that childhood vaccination with thimerosal-containing vac- Conclusion The results do not support a causal relationship between childhood vaccination with thimerosal-containing vaccines and development of autistic-spectrum cines increases the risk of neurodevel- disorders. opmental disorders, such as autism, JAMA. 2003;290:1763-1766 www.jama.com attention-deficit/hyperactivity disorder, and language and speech delay. In a recent independent review con- of autism and other autistic-spectrum has been used in Denmark. The same ducted by the Immunization Safety disorders. vaccine was reformulated without thi- Committee, on behalf of the Institute merosal and used until January 1, 1997, of Medicine, it was concluded that the METHODS when it was replaced with an acellular evidence was inadequate to accept or The Danish childhood vaccination pro- pertussis vaccine.4 The whole-cell vac- reject a causal relationship between thi- gram is voluntary and free of charge to cine was administered at 5 weeks, 9 merosal-containing vaccine and neu- the vaccinees. Vaccines against diph- weeks, and 10 months from 1970 and rodevelopmental disorders.2 How- theria, tetanus, polio, measles, mumps, until it was replaced, irrespective of thi- ever, based on comparison with the rubella, pertussis, and Haemophilus in- merosal content.3 The thimerosal for- toxicology of methylmercury, the bio- fluenzae type b are administered by gen- mulation contained 50 µg of thimero- logical plausibility of a link remained. eral practitioners.3 From 1970, the only sal (∼25 µg of ethylmercury) in the first Further research was recommended. thimerosal-containing vaccine in the We examined the hypothesized asso- program has been the whole-cell per- Author Affiliations: Danish Epidemiology Science Cen- tre, Department of Epidemiology Research (Messrs ciation by comparing children vacci- tussis vaccine. In late March 1992, the Hviid, Wohlfahrt, and Dr Melbye) and Medical De- nated with a thimerosal-containing per- last batch of thimerosal-containing partment (Dr Stellfeld), Statens Serum Institut, Co- penhagen, Denmark. tussis vaccine with children vaccinated whole-cell pertussis vaccine was re- Corresponding Author and Reprints: Anders Hviid, with the same pertussis vaccine formu- leased and distributed from Statens Se- MSc, Danish Epidemiology Science Centre, Depart- ment of Epidemiology Research, Statens Serum Insti- lated without thimerosal and follow- rum Institut. Only the whole-cell vac- tut, Artillerivej 5, DK-2300 Copenhagen S, Denmark ing them with respect to development cine produced by Statens Serum Institut (e-mail: [email protected]).

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dose and 100 µg (∼50 µg of ethylmer- tral Register. From 1995, both inpa- all RRs for age (1-9 years of age,1⁄2-year cury) in each of the succeeding 2 doses. tients and outpatients were included. intervals; 10 years of age, 1-year inter- Since April 1968, all persons in Den- Information on diagnoses of tuberous val) and calendar period (1991-1993, mark have been given a unique identi- sclerosis, Angelman syndrome, fragile 1994, 1995, 1996, 1997, 1998, 1999, fication number in the Danish Civil Reg- X syndrome, and congenital rubella, 2000). We further adjusted our esti- istration System.5 Based on this registry, conditions associated with autism, was mates for the potential confounding we constructed a cohort consisting of all obtained from the National Hospital variables previously listed. Statistical children born in Denmark in the period Discharge Register.8 Information on analysis was performed using PROC from January 1, 1990, to December 31, possible confounding factors was ob- GENMOD in SAS version 6.12 (SAS 1996. Using the unique personal iden- tained from the Danish Civil Registra- Institute Inc, Cary, NC). tification number, we were able to link tion System and the Danish Medical information on vaccinations, diag- Birth Registry,9 as follows: child’s sex, RESULTS noses of autism, diagnoses of other autis- child’s place of birth (Copenhagen, Co- A total of 467450 children were born in tic-spectrum disorders, other relevant penhagen suburbs, area with Ն100000 Denmark between January 1, 1990, and diagnoses, and potential confounders to population, area with population of December 31, 1996. During 2986654 the children in the cohort. The dates of 10000-99999, area with population of person-years of follow-up, we identi- vaccination with 1, 2, or 3 doses of Ͻ10000), birth weight (Ͻ2500, 2500- fied 440 cases of autism and 787 cases whole-cell pertussis vaccine were 2999, 3000-3499, 3500-3999, Ն4000 of other autistic-spectrum disorders. The obtained from the National Board of g), 5-minute Apgar score (0-7, 8-9, 10), mean (SD) age at diagnosis was 4.7 (1.7) Health. We have published details of this gestational age (Ͻ37, 37-41, Ն42 years for autism and 6.0 (1.9) years for process in a study of autistic-spectrum weeks), mother’s age at birth of child other autistic-spectrum disorders. The disorders and measles-mumps-rubella (Ͻ20, 20-24, 25-29, 30-34, 35-39, and follow-up of 5770 children was prema- vaccine.6 Doses administered before June Ն40 years), and mother’s country of turely terminated because of death 1, 1992, were considered to contain thi- birth (Danish or not).The percentage (n=579), emigration (n=5035), disap- merosal, and doses administered after of missing values for the variables birth pearance (n=87), tuberous sclerosis June 1, 1992, were considered thimero- weight, gestational age, 5-minute Ap- (n=51), Angelman syndrome (n=17), sal-free. Children who received thimero- gar score, mother’s country of birth, and or congenital rubella (n=1). sal-free vaccine after 1 or 2 doses of thi- child’s place of birth were 6.6%, 6.9%, In our cohort, only 20755 (4.4%) merosal-containing vaccine were 6.9%, 0.3%, and 0.03%, respectively. children did not receive any whole- classified only according to receipt of thi- Children in our cohort contributed cell pertussis vaccine, 446695 (95.6%) merosal-containing vaccine. person-time to follow-up from 1 year of were vaccinated at least once, 416081 Information on autism and other au- age or January 1, 1991, whichever (89.0%) were vaccinated twice, and tistic-spectrum disorder diagnoses was occurred last, until a diagnosis of autism, 293186 (62.7%) received 3 doses of obtained from the Danish Psychiatric other autistic-spectrum disorder, tuber- whole-cell pertussis vaccine. Among Central Register.6,7 Child psychiatrists ous sclerosis, Angelman syndrome, frag- those who received at least 1 thimero- make the diagnosis and assign diagnos- ile X syndrome or congenital rubella, sal-containing pertussis vaccine tic codes for this register. In the pe- possible death, disappearance or emi- (n=138953), 118593 received 1 sub- riod 1991-1993, the International Clas- gration, 11 years of age, or until Decem- sequent dose and 65725 received 2 sub- sification of Diseases, 8th Revision ber 31, 2000, whichever occurred first. sequent doses of thimerosal-contain- (ICD-8) was used. In the period 1994 Follow-up was begun at 1 year of age ing vaccine. Furthermore, 42032 through 2000, the International Clas- because indications for an evaluation of children who received at least 1 dose sification of Diseases, 10th Revision (ICD- a possible case of autistic-spectrum dis- of thimerosal-containing vaccine sub- 10) was used. All cases of autism and order typically occur after the first year sequently received at least 1 dose of thi- other autistic-spectrum disorders in our of life. The resulting incidence rates for merosal-free vaccine. In those receiv- study have been ascertained using ICD- autism and other autistic-spectrum dis- ing at least 1 dose of whole-cell pertussis 10. Autism was defined by ICD-10 code orders were analyzed with Poisson vaccine, there were 407 cases of au- F84.0, which is similar to Diagnostic and regression, producing estimates of rate tism (303 receiving thimerosal-free and Statistical Manual of Mental Disorders, ratios (RRs) according to vaccination his- 104 receiving thimerosal-containing Fourth Edition (DSM-IV) code 299.00, tory.10 Vaccination history was consid- vaccine) and 751 cases of other autistic- and other autistic-spectrum disorders ered a time-varying variable. We esti- spectrum disorders (430 receiving thi- were defined by ICD-10 codes F84.1- mated the dose-response relationship merosal-free and 321 receiving thi- F84.9, which are similar to DSM-IV between thimerosal-containing vac- merosal-containing vaccine). codes 299.10 and 299.80. cine and autism and other autistic- Comparing children vaccinated with In 1991-1994, only inpatients were spectrum disorders as the increase in RR at least 1 dose of thimerosal-contain- included in the Danish Psychiatric Cen- per 25 µg of ethylmercury. We adjusted ing whole-cell pertussis vaccine with

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children vaccinated with a thimerosal- ing our cohort to children born in 1991- 1, 1995, to December 31, 2000, a pe- free formulation of the same vaccine, 1993, a presumably more homoge- riod where outpatients were included, we found a fully adjusted RR of 0.85 neous group with respect to diagnosis, we found similar trends (RR, 1.24 [95% (95% confidence interval [CI], 0.60- length of follow-up, and factors not in- CI, 1.16-1.32] for autism; RR, 1.20 1.20) for autism and an RR of 1.12 (95% cluded in this study (eg, mercury ex- [95% CI, 1.13-1.26] for other autistic- CI, 0.88-1.43) for other autistic- posure through food) and found a fully spectrum disorders). spectrum disorders (TABLE). Further- adjusted RR of 0.86 (95% CI, 0.53- more, we found no evidence of a dose- 1.39) for autism and an RR of 1.05 (95% COMMENT response association between the dose CI, 0.77-1.44) for other autistic- We found no evidence of an associa- of ethylmercury received and autistic- spectrum disorders and no evidence of tion between thimerosal-containing vac- spectrum disorders (increase in RR per a dose-response association (increase cine and autism in children who 25 µg of ethylmercury, 0.98 [95% CI, in RR per 25 µg of ethylmercury, 0.97 received thimerosal-containing vac- 0.90-1.06] for autism and 1.03 [95% CI, [95% CI, 0.85-1.10] for autism and 1.04 cine compared with children who 0.98-1.09] for other autistic-spectrum [95% CI, 0.96-1.13] for other autistic- received the same vaccine formulated disorders). spectrum disorders). without thimerosal. Furthermore, there Although doses administered after Finally, we evaluated the impact of was no indication of a dose-response June 1, 1992, were considered thimero- missing values by the method of single association between autism and the sal-free, it is conceivable that a few thi- imputation, replacing a missing value amount of ethylmercury received merosal-containing doses may have been with the most common value of the through thimerosal. administered during the months after relevant variable, and found a fully The hypothesis of an association this date. To assess whether misclassi- adjusted RR of 0.85 (95% CI, 0.60- between thimerosal and autism has pri- fication of vaccine type might have bi- 1.20) for autism and 1.13 (95% CI, marily been based on biological plau- ased our estimates, we reestimated the 0.89-1.44) for other autistic-spectrum sibility through analogies with meth- RRs, omitting children vaccinated from disorders. ylmercury.2 Ethylmercury, however, is June 1, 1992, through December 31, To evaluate whether the incidence of thought to have a shorter half-life in the 1992. We found a fully adjusted RR of autistic-spectrum disorders was increas- human body than methylmercury, and 0.87 (95% CI, 0.61-1.23) for autism and ing in Denmark in the study period, we no controlled studies of low-dose eth- an RR of 1.15 (95% CI, 0.90-1.47) for calculated time period trends from our ylmercury toxicity in humans have been other autistic-spectrum disorders and no cohort. We found statistically signifi- conducted.11 Pichichero and col- evidence of a dose-response associa- cant increases in age-adjusted RR per cal- leagues12 measured the concentration tion (increase in RR per 25 µg of ethyl- endar year for both autism and other au- of mercury in the blood, urine, and stool mercury, 0.98 [95% CI, 0.90-1.07] for tistic-spectrum disorders during the of infants who received thimerosal- autism and 1.04 [95% CI, 0.98-1.09] for study period (RR, 1.24 [95% CI, 1.17- containing vaccines and concluded that other autistic-spectrum disorders). 1.31] for autism; RR, 1.21 [95% CI, 1.16- vaccination did not raise the blood con- In a further analysis we evaluated the 1.27] for other autistic-spectrum centration of mercury above safe lim- robustness of our results by restrict- disorders). In the period from January its, and that ethylmercury was rapidly

Table. Rate Ratio of Autism and Other Autistic-Spectrum Disorders Comparing Children Vaccinated With a Thimerosal-Containing Vaccine to Children Vaccinated With a Thimerosal-Free Formulation of the Same Vaccine Autism Other Autistic-Spectrum Disorders

Person-Years No. of No. of at Risk Cases RR (95% CI)* RR (95% CI)† Cases RR (95% CI)* RR (95% CI)† Vaccinations All thimerosal-free 1 660 159 303 1.00 1.00 430 1.00 1.00 Any containing thimerosal 1 220 006 104 0.85 (0.60-1.20) 0.85 (0.60-1.20) 321 1.12 (0.88-1.43) 1.12 (0.88-1.43) Doses of thimerosal-containing vaccine None 1 660 159 303 1.00 1.00 430 1.00 1.00 1 dose (25 µg eHg) 169 920 18 0.99 (0.59-1.68) 1.01 (0.60-1.71) 40 0.96 (0.67-1.39) 0.95 (0.66-1.37) 2 doses (75 µg eHg) 447 973 33 0.71 (0.46-1.09) 0.70 (0.46-1.09) 130 1.20 (0.92-1.56) 1.20 (0.92-1.56) 3 doses (125 µg eHg) 602 113 53 0.96 (0.63-1.46) 0.96 (0.63-1.47) 151 1.11 (0.83-1.48) 1.13 (0.84-1.51) Trend (increase in RR 0.98 (0.90-1.06) 0.98 (0.90-1.06) 1.03 (0.97-1.09) 1.03 (0.98-1.09) per 25 µg eHg) Abbreviations: CI, confidence interval; eHg, ethylmercury; RR, rate ratio. *Adjusted for confounders: age and calendar period. †Fully adjusted: age, calendar period, child’s sex, child’s place of birth, birth weight, 5-minute Apgar score, gestational age, mother’s age at birth of child, and mother’s country of birth.

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eliminated via the stools. They esti- Madsen and colleagues6 found Danish der can be a lengthy process; this is re- mated the blood half-life of ethylmer- prevalence rates for autism and other flected in the mean ages of diagnoses cury at 7 days (95% CI, 4-10 days), al- autistic-spectrum disorders compa- in this study (4.7 years for autism and though their study was not designed as rable to prevalence rates found in other 6.0 years for other autistic-spectrum a formal pharmacokinetic study of eth- studies.Thus we conclude that the va- disorders). However, this is more likely ylmercury. lidity and completeness of the autism to be a problem in an incidence study In 1999, when thimerosal was still and other autistic-spectrum disorder di- than in a risk factor study. widely used, children in the US child- agnoses in the Danish Psychiatric Cen- In conclusion, our results are not hood immunization program would tral Register is high. However, it is pos- compatible with the hypothesis of a have received 187.5 µg of ethylmer- sible that the National Hospital causal association between thimerosal cury by the age of 6 months and 237.5 Discharge Register is not complete with and autistic-spectrum disorders. µg of ethylmercury by the age of 2 respect to a diagnosis of tuberous scle- 2 years. In Denmark, children would rosis, Angelman syndrome, fragile X Author Contributions: Study concept and design: have received 125 µg of ethylmercury syndrome, and congenital rubella. How- Hviid, Stellfeld, Wohlfahrt, Melbye. Analysis and interpretation of data: Hviid, Wohlfahrt. by the age of 10 months. However, in ever, these conditions are rare in the Drafting of the manuscript: Hviid. the Danish program, children re- general population and since we have Critical revision of the manuscript for important in- tellectual content: Stellfeld, Wohlfahrt, Melbye. ceived larger doses of ethylmercury per compared only vaccinated children, Statistical expertise: Hviid, Wohlfahrt. vaccine (50 µg compared with 25 µg in lack of completeness is unlikely to se- Obtained funding: Hviid, Melbye. the United States) so that at 3 months, riously confound an association be- Study supervision: Wohlfahrt, Melbye. Funding/Support: This study was supported by grant Danish children would have received tween thimerosal content and autistic- 11 from the Danish National Research Foundation and the same amount of ethylmercury as US spectrum disorder. grant 22-02-0293 from the Danish Medical Re- search Council. children (75 µg).2 We found statistically significant in- To our knowledge, our study is the creased rates over time for both autism first population-based cohort study to and other autistic-spectrum disorders. REFERENCES examine the association between thi- These results are compatible with a dra- 1. Clarkson TW. The toxicology of mercury. Crit Rev merosal and autism. In Denmark since matic increase in the number of diag- Clin Lab Sci. 1997;34:369-403. 2. Stratton K, Gable A, McCormick MC, eds. Immu- 1970, only the whole-cell pertussis vac- nosed cases of autistic-spectrum disor- nization Safety Review: Thimerosal-Containing Vac- cine was formulated with thimerosal, ders during the study period, similar to cines and Neurodevelopmental Disorders. Washing- ton, DC: National Academy Press; 2001. and this vaccine was the only one used what has been observed in other coun- 3. Plesner AM, Ronne T. The childhood vaccination for pertussis immunization until it was tries (eg, the United States). program: background, status and future [in Danish]. Ugeskr Laeger. 1994;156:7497-7503. replaced with an acellular pertussis vac- In Denmark, general practitioners ad- 4. Ronne T. The Danish vaccination program for chil- cine in 1997. The unique situation has minister all childhood vaccinations and dren [in Danish]. Ugeskr Laeger. 1997;159:1584- allowed a direct comparison of chil- are reimbursed when reporting these to 1585. 5. Malig C. The Civil Registration System in Den- dren vaccinated with a thimerosal- the National Board of Health, thus en- mark. Bethesda, Md: International Institute for Vital containing whole-cell pertussis vac- suring a high degree of completeness. Registration and Statistics; 1996. IIVRS Technical Pa- per No. 6. cine with children vaccinated with the In our cohort we found that 96%, 89%, 6. Madsen KM, Hviid A, Vestergaard M, et al. A popu- same vaccine formulated without thi- and 63% of children were vaccinated at lation-based study of measles, mumps, and rubella vaccination and autism. N Engl J Med. 2002;347:1477- merosal, and thus we have avoided con- least once, at least twice, and 3 times 1482. founding by contraindication and other with whole-cell pertussis vaccine. The 7. Munk-Jorgensen P, Mortensen PB. The Danish Psy- selection bias associated with unvacci- low uptake of 3 doses is unexpected but chiatric Central Register. Dan Med Bull. 1997;44:82- 84. nated children. Furthermore, we have can be partially explained by the tran- 8. Andersen TF, Madsen M, Jorgensen J, Mellemk- no reason to believe that the 2 groups sition to acellular pertussis vaccine in joer L, Olsen JH. The Danish National Hospital Reg- ister: a valuable source of data for modern health sci- of children differ with respect to other January 1997. Furthermore, for each ences. Dan Med Bull. 1999;46:263-268. potential risk factors for autism. dose there is a small chance of either 9. Knudsen LB, Olsen J. The Danish Medical Birth Reg- istry. Dan Med Bull. 1998;45:320-323. All data used in this study were col- missing the dose or the vaccination not 10. Clayton D, Hills M. Statistical Models in Epide- lected prospectively, eliminating con- being recorded. Even small probabili- miology. Oxford, England: Oxford University Press; 1993. cerns about recall bias. Madsen and col- ties for each dose can, if they are sta- 11. Ball LK, Ball R, Pratt RD. An assessment of thi- 6 leagues reviewed the medical records tistically independent, result in a sig- merosal use in childhood vaccines. Pediatrics. 2001; of 40 children with autism from the nificant reduction in the calculated 107:1147-1154. 12. Pichichero ME, Cernichiari E, Lopreiato J, Treanor Danish Psychiatric Central Register and uptake of all 3 doses. J. Mercury concentrations and metabolism in infants found that 37 children met the opera- A possible weakness of this study is receiving vaccines containing thimerosal: a descrip- tive study. Lancet. 2002;360:1737-1741. tional criteria for autism according to that the date of diagnosis used as the 13. Bertrand J, Mars A, Boyle C, Bove F, Yeargin- a systematic coding scheme devel- incidence date may differ significantly Allsopp M, Decoufle P. Prevalence of autism in a United States population: the Brick Township, New oped by the Centers for Disease Con- from the “onset of symptoms” date. A Jersey, investigation. Pediatrics. 2001;108:1155- trol and Prevention.13 Furthermore, diagnosis of autistic-spectrum disor- 1161.

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