34 Lines of Evidence: Read the evidence for yourself

Pharmacological/toxicological evidence

Evidence / references

Background / additional information

1. Toxic Metabolite: Mechanisms of APAP-mediated injury are plausible. The body converts a fraction of acetaminophen into a toxic metabolite called NAPQI. For review, see Jones et al. [1]  

The first study showing that children with ASD are deficient in a metabolic pathway necessary to safely detoxify APAP in babies (sulfation) is now more than a quarter of a century old [2], and was subsequently corroborated [3, 4].

2. Oxidative Stress: Genetic, epigenetic, and environmental factors associated with an increased risk of ASD are also associated with oxidative stress, which has an adverse effect on the body’s ability to safely metabolize APAP [2, 5, 6].

The wide array of factors associated with ASD have led to the hypothesis that many things can come together to cause ASD, but ASD is characterized by impairment of social function and other particular behavioral phenotypes, suggesting specificity in the etiology of the condition.

3. One enzyme (CyP450 2E1) which produces the toxic metabolite of APAP (NAPQI) is expressed in the human brain from before birth [7], is a target of epigenetic alterations in mothers who have children with ASD [8] and in mothers who took APAP during labor and delivery [9].

Polymorphisms in another enzyme (CyP450 1A2) that produces the same toxic metabolite of APAP is associated with ASD [10, 11].

4. Phase II metabolism is the way the body processes APAP without producing the toxic metabolite. The first study showing that children with ASD are deficient in a phase II metabolic pathway necessary to safely detoxify APAP in babies (sulfation) is now more than a quarter of a century old [2], and was subsequently corroborated [3, 4].

Besides sulfation, the human body has one other primary metabolic pathway to metabolize APAP without producing the toxic metabolite, NAPQI. That pathway, glucuronidation, is also apparently deficient in children with ASD [12].

5. Toxicity in cats: APAP is not used in domestic cats because they lack of a robust glucuronidation-dependent capacity for metabolism [13-17], making them susceptible to APAP-mediated injury. Human neonates also lack a robust glucuronidation-dependent pathway [18, 19].

Based on liver function in human babies and children, APAP was incorrectly determined to be safe for pediatric use in the 1960s and 1970s (see line of evidence #20 in Table 4), before this evidence became available in the 1980s.

6. Increasing evidence suggests that the gut/brain axis may play a role in many cases of ASD (Reviewed recently [20, 21]), and APAP is known to adversely affect gut function in laboratory animal models [22-24] and possibly in humans [25].

Aberrant gut function leading to oxidative stress and inflammation is among many factors that would predispose individuals to adverse reactions to APAP leading to ASD [6], and gut microbial metabolites serve as excellent biomarkers for ASD [26, 27]. However, it remains unknown whether aberrant gut function can be induced by APAP at the time of ASD induction and play a role in that induction.

7. APAP binds directly to arachidonic acid [28] and affects arachidonic acid metabolism [29]. Alterations of arachidonic acid [30] and enzymes involved in arachidonic acid metabolism [31, 32] are associated with ASD.

It is unknown what role arachidonic acid plays in ASD, but arachidonic acid plays a role in both the analgesic and antipyretic properties of APAP, and its metabolism is associated with ASD.

8. Decreased phase II metabolism at birth: Human neonates lack a robust glucuronidation-dependent pathway [18, 19], one of the two main metabolic pathways that avoids the production of NAPQI, the toxic metabolite of APAP.

Although individuals with ASD are particularly deficient in both main pathways used to metabolize APAP without producing the toxic metabolite (sulfation and glucuronidation), all individuals are deficient in glucuronidation at the time of birth. It is during this time period (labor and delivery) when the greatest risks of ASD associated with APAP exposure are found [33].

9. Mitochondrial function may be impaired in individuals with ASD [34], and aberrant metabolism of APAP can impair mitochondrial function [35]. Calcium signaling is known to be important in ASD [36], and APAP adversely affects calcium signaling [37]. 

Calcium signaling dysfunction can be used as a biomarker for ASD [38], although it remains unknown whether APAP plays any role in inducing that dysfunction.

10. Hormesis is the toxicological principle in which exposure to a relatively low dose of toxin can result in protection from exposure to the same or even other toxins later in life. Evidence indicates that low levels of exposure to ethanol [39-42] and perhaps APAP [43, 44] during pregnancy can protect an individual from ASD, suggesting that exposure to toxin later in life (after pregnancy) may be critical in the etiology of ASD.

The observation of an apparent hormetic effect of alcohol and acetaminophen during pregnancy related to ASD is not (with emphasis) considered a justifiable reason for intentional exposure to toxins during pregnancy. Rather, it is an indication that neurodevelopmental toxins (e.g., acetaminophen) should be avoided after pregnancy. Alcohol exposure during pregnancy promotes accelerated liver development in laboratory animals [45], and exposure to acetaminophen can increase resistance to future acetaminophen toxicity [46], suggesting that potential hormetic effects of alcohol and acetaminophen exposure during pregnancy, as related to ASD, are plausible.

Associations with Medical Practice/APAP use

Evidence / references

Background / additional information

11. Circumcision of males was associated with a 2-fold increase in the risk for early-onset (infantile) ASD in Danish children born from 1994 to 2003 [47]. Further, a study of US males (56% of participants born from 1982 to 1996, when acetaminophen use in infants would likely have been favored over aspirin) identified social problems associated with circumcision [48]. APAP is sometimes used as an analgesic following circumcision despite the fact that such use is of highly questionable effectiveness [49]. However, administration of APAP by physicians and by parents following infant circumcision has not been widely documented.

A study of US males born between 2003 and 2023 found no correlation between circumcision and the prevalence of ASD [50]. Although the US study was substantially less powered than the earlier Danish study [47], the potential effects of acetaminophen exposure as a result of circumcision seen in the Danish boys are likely to be overshadowed or “swamped out” in other populations by the presence of a wide range of factors that increase the use of acetaminophen (see line of evidence #31) [51]. Increases in infant vaccine administration are of particular significance, given that inflammation-induced oxidative stress is a cofactor in the APAP-mediated induction of ASD [6]; Acetaminophen exposure following a potent inflammatory stimulus (e.g., an infection or a vaccine) is expected to carry more risk than acetaminophen exposure for reasons not involving a potent inflammatory stimulus (e.g., uncomplicated circumcision or typical teething). Consistent with this view, studies in both laboratory animals [52] and in children [53] indicate that infections enhance the toxicity of acetaminophen. 

12a. Epidemiology (pregnancy): Use of APAP during pregnancy has been associated with adverse long-term effects on the mental health of offspring in numerous studies  [33, 54-76].

This line of evidence has received more attention than any other line of evidence, to the point of being the only line of evidence considered by many investigators. However, the numerous studies underpinning this line of evidence are hampered by several factors which can cause errors in estimation of the association between APAP and ASD [1]. For example, a recent study found a dramatic association (odds ratio (OR) for ASD with APAP use = 1.8) [77], but incorrectly and completely cancelled out that association using an error in the assumptions underlying the statistical analysis [1, 76, 78].

12b. Epidemiology (childhood): Analysis of the Danish National Birth Cohort (DNBC) by Alemany and colleagues revealed an odds ratio (OR) of 1.3 (CI 1.02–1.66) for ASD associated with postnatal APAP exposure [59], despite the fact that the use of APAP appears to be dramatically underreported in the DNBC [79].

Alemany and colleagues averaged the results from the DNBC with assessments of autism-like symptoms (not ASD) from smaller data sets, and reported no association between postpartum APAP use and those symptoms (not ASD) in the abstract of the paper [59]. This issue has been addressed in detail by us in the literature [78, 79], but unfortunately may still result in confusion [80]. In addition, the study [59] employed invalid statistical adjustments expected to underestimate the association between APAP and ASD [1, 78]. See text for additional discussion. In a study without the problems cited above, Bittker and Bell found a 2.3% (CI 0.5 % - 4.3 %) greater risk of ASD in male children associated with each administration of APAP between birth and two years of age [81].

13. Regression: APAP use during early childhood is associated with a 20-fold greater risk of regressive ASD [82]. APAP use with mild adverse reactions to a vaccine, but not mild adverse reactions to a vaccine alone, is associated with ASD [82].

This case-controlled study, now more than 16 years old, has been widely criticized, but careful analysis does not reveal any credible objections [79]. This study was the first study to separate the impact of vaccines from APAP on neurodevelopment, and the first to implicate APAP with the etiology of ASD.

14. Higher levels of APAP in cord blood are associated with ASD [33].

For the analysis, the authors divided the women into three groups based on cord blood APAP levels. The third with the highest levels had 3.6 times more likelihood of having a child with ASD than the third with the lowest levels of APAP.

15. Ultra-Orthodox Jews [83] in Israel have a reported prevalence of ASD less than half of that of reform Jews. Traditional circumcision practices employed by Ultra-Orthodox Jews do not utilize APAP. Further, Orthodox Jews may use less pain medications during childbirth than others [84].

Circumcision is often performed using APAP as an analgesic despite the fact that such use is of highly questionable effectiveness [49]. Almost all Israeli Jews are circumcised [85]. Further, decreased use of pain medications during labor and delivery, which may be associated with Orthodox Judaism [84], would likely lower exposure to APAP at the time of life when individuals are the most sensitive to APAP-mediated neurodevelopmental injury [86].

Laboratory animal studies

Evidence / references

Background / additional information

16. Numerous studies in laboratory animals from multiple laboratories indicate that early life exposure to APAP causes long term changes in brain function [80, 87-101].

After adjusting for weight, the amount of APAP that causes profound changes in laboratory animals in some studies is very close to [97] or even less than [96] the amount administered to human babies and children. Thus, APAP could never be used in babies or children if current guidelines for drug safety were applied.

17. Early life exposure to APAP has a greater long-term impact on male laboratory animals than female laboratory animals [92, 99, 102, 103]. ASD is more common in males than in females.

The reason or reasons why males are more susceptible to APAP-mediated injury has been considered in some detail, and several plausible mechanisms have been proposed [99, 102].

18. Prenatal exposure of laboratory rats to APAP causes problems with the processing of sound [80] and with olfactory function [95]. Impairment of olfactory function [104] and some degree of auditory dysfunction (reviewed by Graeca and Kulesza [80]) are associated with ASD.

It is unknown whether these effects of APAP on laboratory animals are related to altered processing of smell and hearing in some individuals with ASD.

19. APAP causes apoptosis-mediated death of cortical neurons in laboratory rats [105], and cortical neurons may be involved in the pathology of ASD [106, 107].

Increased levels of biomarkers for neuronal apoptosis [108-110] and impaired autophagy [111] are associated with ASD. Autophagy is necessary to clearing damaged organelles such as mitochondria [112], which are created by aberrant metabolism of APAP [35].

Miscellaneous observations

Evidence / references

Background / additional information

20. APAP was never demonstrated to be safe for neurodevelopment [113]. Over two hundred papers in the medical literature claim that APAP is safe for babies and/or children when used as directed, but all studies were based on the false assumption that adverse reactions in babies would involve easily measured liver injury, the same as in adults [113].

One study in laboratory animals in the 1980s showed that even lethal doses of APAP do not cause liver failure in neonates [114], but the first study showing APAP-mediated neurodevelopmental brain injury in laboratory animals was not published until 2013 [97].

Like APAP, opioids have also never been shown to be safe for neurodevelopment [115]. However, unlike APAP, opioids are not generally assumed to be safe for neurodevelopment when used as directed. Further, one study probing the safety of prenatal opioid exposure found reductions in communication skills in children associated with prenatal APAP exposure, but not with prenatal opioid exposure [60].

21. APAP temporarily blunts social trust [116] and awareness [117], emotional responses to external stimuli [118], and the ability to identify errors [119] in adults.

Although the mechanisms are unknown, these studies show that APAP affects aspects of mental function that are impaired in individuals with ASD.

22. APAP inhibits neuronal cell growth in tissue culture experiments, altering “arborization”, the process by which neurons branch out to make connections with other neurons [120]. APAP [105, 121] and a metabolite of APAP [122] also cause death of brain cells in culture.

Adverse effects of APAP on neuronal cell growth in culture (in vitro) are dose dependent, and observable at concentrations near those achieved in clinical therapy [120]. These effects in vitro would discourage use of APAP in humans if current guidelines for drug safety were applied.

23. Observations of parents: The top three conditions identified as a cause of their child’s regression into autism listed by parents were vaccination (51% of parents), high fever (11% of parents), and illness (8% of parents)[123]. A standard treatment for all of these conditions is APAP. Surveys show that up to 50% of parents who have a child with ASD believe that their children’s ASD was induced by a vaccine [124, 125].

Although parent’s belief in the connection between vaccination and ASD has been widely attributed to a 1998 case series describing an association between the MMR vaccine and autism in 12 patients [126], the title of that report is not intelligible to individuals outside of the medical profession, and medical papers have seldom affected public opinion. A more likely explanation involves the induction of ASD by APAP use concurrent with vaccination, as suggested by Schultz [82, 127]. The failure to consider seriously the views of parents has historically been problematic, resulted in two system-wide errors that were influential for decades, the refrigerator mother hypothesis and the failure to recognize regression [6].

24. Cystic fibrosis (CF) is associated with unusually efficient (effective) metabolism of APAP [128, 129], and the prevalence of ASD is apparently very low in patients with cystic fibrosis [6].

The mental health of patients with cystic fibrosis has been characterized extensively, but no association between ASD and cystic fibrosis has been reported.

25. The “missing heritability” paradox of ASD suggests that epigenetic factors or very early exposure to environmental factors might influence the onset of ASD [130].

The role of APAP in the induction of ASD nicely resolves the missing heritability paradox connected with ASD, in which sibling studies indicate a high contribution of genetics, but genome wide studies fail to identify the genes involved [130]. The observation that abuse of a mother when she was a child is associated with ASD in the offspring [131] is one example of evidence that supports this view.

26. ASD and fetal alcohol spectrum disorder (FASD) are similar in many regards. Reviewed by Jones et al. [1] A spectrum disorder can also be triggered by the drug valproate [132].

These observations demonstrate that a complex spectrum disorder (FASD) sharing many similarities with ASD can (a) be induced by a single chemical and (b) be influenced by a variety of genetic and environmental factors.

27. Common alternative explanations are not consistent with known observations and/or require elaborate/complex scenarios to be true.

Some alternative explanations depend on the view that the incidence of ASD has not increased dramatically over time, a view that is contradicted by numerous lines of evidence showing that ASD is an effect of industrialized culture. Evidence that rules out alternative explanations for the etiology of ASD have been described previously [1, 79].

28. APAP exposure in utero is associated with increases in childhood brain cancer. This connection was first noted in 2010 in Sweden using a case-controlled method [133], although the association was not statistically significant (OR 1.7; CI 0.6–5.4). A more recent study from Taiwan [134] supported the association and was statistically significant (HR 2.4; CI 1.10–5.39).

In the study from Taiwan, which evaluated cancer in about 2.27 million mother-child pairs [134], medulloblastoma, a type of brain cancer, was the only type of cancer in children significantly associated with prenatal use of APAP. An increased risk of cancer might potentially result of cellular injury and subsequent repair of that injury [135], although it remains unknown if APAP-associated brain cancer risks are related to processes involved in the etiology of ASD.

Associations in place and time

Evidence / references

Background / additional information

29. The incidence of ASD began to increase in the early 1980s, coinciding with the increase in APAP use after aspirin was associated with Reye’s syndrome [6]. The ratio of regressive to infantile ASD also rose at the same time [136]. 

Temporal associations do not prove causality, but are a necessary prerequisite for causality to exist. The increased ratio of regressive to infantile ASD, noted in 2000 [136], would suggest that something was introduced into the environment that could induce ASD after months or even years of neurodevelopment. This factor was tragically and incorrectly suspected to be a vaccine at that time, an issue that was decisively addressed by Stephen Schultz eight years later (see line of evidence #13 in Table 2).

30. The popularity of APAP use and the prevalence of ASD was substantially higher in Denmark than in Finland in the mid-2000s [86].

Geographic-dependent associations do not prove causality, but do contribute to the total body of evidence. Particularly in the absence of alternative explanations, these associations can be compelling.

31. The incidence of ASD has steadily increased [6] as direct-to-consumer advertising [137] and marketing of over-the-counter medications encouraged APAP exposure early in life.

Temporal associations do not prove causality, but are a necessary prerequisite for causality to exist. Alternative explanations for the rise in prevalence of ASD face several insurmountable problems, previously reviewed [1, 79]. One possible explanation for the persistence of unrealistic alternative explanations may be that many investigators are unaware of a satisfactory explanation consistent with available evidence.

32. Factors other than the move away from aspirin and advertising have led to increasing use of APAP that corresponds to increasing levels of ASD [51]. For example, many parents use APAP to avoid or treat adverse reactions to vaccines [138, 139], and the vaccine schedule has dramatically increased since 1980. In addition, use of APAP during labor and delivery [140-142] and in the NICU [143] have increased as a result of APAP use for ERAS protocols developed to reduce opioid exposure after the opioid pandemic of the 2010s.

Emerging data are consistent with a dramatic rise in the prevalence of ASD among subpopulations with low socioeconomic status [50, 51, 144]. Evidence suggests that an adverse reaction between acetaminophen and cannabis use disorder (CUD) may underlie this apparent phenomenon [145]. Lethal drug-drug interactions between cannabis and APAP have been demonstrated in a laboratory animal model [146], and APAP and cannabis interact with the same pathways in the brain to mediate their respective therapeutic effects. Thus, although heavy cannabis exposure is not expected to dramatically increase the use of APAP, it could increase the adverse impact of APAP on neurodevelopment, providing a plausible explanation for socioeconomic-depending trends in ASD prevalence [51, 145].

33. Numerous investigators noted a very low prevalence of ASD prior to 1970 or noted increases in the prevalence of ASD that could not be attributed to factors other than a real increase. Reviewed by Jones et al. [1]

Grunya Sukhareva [147, 148] and Leo Kanner [149, 150] were both renowned for their breadth of knowledge and grasp of pediatric psychology, and both described autism essentially the same as it is described today. Yet neither recognized a high prevalence of the disorder in the 1920s and 1940s, respectively. Further, neither was ridiculed by contemporaries for describing a common, previously well-known condition.

34. Studies in several countries with chronic shortages of medication found dramatically lower-than-expected levels of ASD relative to other developmental issues, including Down syndrome. Reviewed by Jones et al. [1] 

In addition to evidence presented in the previous review of this issue [1], apparently low levels of ASD in Cuba have been identified, where 241 cases of ASD in the entire nation (1 in 25,000 children) were identified in a 2016 report [151]. APAP is available in Cuba by prescription only [152], and multiple travel advisors cite APAP in particular as being in short supply in Cuba [153-156]. In addition, individuals with pervasive developmental disorders, about 50% of which typically have ASD, were found among second generation Israeli immigrants from Ethiopia, but not among first generation Israeli immigrants from Ethiopia (p = 0.0022) [157].