Smaller doses act differently to larger ones
Dr Clare Craig
| Key points • The same molecule at different doses can produce different effects and sometimes opposite ones. • Regulators expect different efficacy by dose including non-linear dose responses • Safety assessment is built around finding the dose at which harm begins as the dose rises. • The folic acid safety signals are seen below 1 mg, which is the range fortification will deliver. The trials claiming safety at 4 mg cannot be used to dismiss them. • Lifelong repeated low dose exposure through food has never been tested. |
In 1983, in Science Ian Zagon and Patricia McLaughlin published a result that should have transformed safety testing. They gave naltrexone, a drug that blocks opioid receptors and is used to treat opioid and alcohol dependence, to mice inoculated with cells from a cancer called a neuroblastoma which every untreated mouse went on to develop. There were three key observations: the percentage who developed a tumour; the time before a tumour developed and the percentage that survived the experiment. At the low dose of 0.1 milligrams per kilogram, a third developed the tumour; the time before a tumour appeared nearly doubled and overall survival rose by 36 per cent. At a much higher dose of 10 milligrams per kilogram, every mouse developed a tumour, tumours appeared sooner than in the untreated controls and survival fell by 19 per cent. Conventional medical thinking that more drug has more effect should have been abandoned that day. Here was a result showing that the same molecule, in the same strain of mouse had the opposite effect on the same tumour depending entirely on the dose.
The following year the same authors discovered why. The low dose only blocked opioid receptors for four to six hours a day while the high dose blocked them continuously. The intermittent blockade led to a rebound response in the animal’s own opioid signalling which restrained tumour growth. However, continuous blockade removed that signalling altogether. The dose mattered because of the timing of its effect on the target receptor.
The low dose is now prescribed off licence as a treatment in its own right for its anti-inflammatory effects, and its anti-cancer potential is under investigation on the strength of results like these. The high dose does not help in these scenarios. The two doses are pharmacologically unique in their actions. They are not just proportional effects of a higher dose.
Folic acid is very similar in terms of dosage effects. In the randomised trial evidence cancer incidence was increased in trials of less than one milligram a day. However, at doses over one milligram there is no cancer signal. In addition, in the only large trial of a modest dose in women at ordinary risk of having a baby with a neural tube defect, there was an excess of fetal deaths. The trials at four milligrams did not show a significant excess, though the largest of them was not able to rule one out. The lack of a signal at higher doses is used to dismiss the signal seen at lower doses. This is not good science. It is also terrible public health when the entire country is about to be exposed on a daily basis to the lower dose.
Different efficacy at different doses
Naltrexone is not a one-off. There are numerous drugs which show different effects depending on doses.
| Drug | Lower dose | Higher dose |
| Aspirin | 75 mg daily, to prevent arterial clotting | 300 to 900 mg for pain, several grams a day for inflammatory disease |
| Amitriptyline | 10 to 25 mg at night, for neuropathic pain and migraine prevention | 75 to 150 mg and above, for depression |
| Doxycycline | 20 mg twice daily for periodontitis, below the antibacterial concentration | 100 to 200 mg daily, as an antibiotic |
| Methotrexate | 7.5 to 25 mg once a week, for rheumatoid arthritis and psoriasis | Multiple grams for leukaemia and osteosarcoma |
| Naltrexone | 1.5 to 4.5 mg, used off licence for inflammatory and pain conditions | 50 mg, for opioid and alcohol dependence |
For example, doxycycline is an antibiotic but it has an unrelated indication. At 20 milligrams twice a day it cannot inhibit bacteria but does dampen collagen-degrading enzymes so it is licensed for reducing gum disease. Trials at that dose have found no detectable antibacterial effect and therefore no emergence of resistant organisms.
Quetiapine is an example where there are more than two doses of interest. At 25 milligrams the prominent action is blockade of histamine receptors causing sedation. At higher doses serotonin receptors are occupied in the brain, although this is not used as a treatment. Once we reach several hundred milligram doses dopamine receptors are occupied and it can be used to treat psychosis. The molecule stayed the same but there was one of three outcomes in terms of effects in the body based only on dosage.
These are quite separate treatments that happen to use the same molecule.
Why can dosage have such a huge effect?
Bodies are complex systems. Drugs tend to interact by binding to receptors on cells. There are often multiple potential binding sites in the body and they differ in how tightly the drug binds to them. At low concentration only sites with the strongest binding are occupied. As the concentration rises the molecule binds to other sites where binding is less strong and the resulting effect that predominates therefore changes.
Rather than think of drugs as having a linear reaction on the body the effect is the sum of everything the molecule is doing at every binding site it reaches, combined with whatever the body does in response. The body is full of buffering systems such that when a threshold is reached the response changes. Therefore, different dosages can lead to entirely different responses.
All of that is accepted as true when assessing drug efficacy. There is a guideline which sets the standard: E4 of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, adopted by the regulators of Europe, Japan and the United States in 1994. This sets out what must be known about dosage before a medicine can be registered. E4 does not assume that increasing doses increases the benefit. It recommends that study designs look to see whether lower doses are in fact more efficacious than higher ones.
Toxicology is a field which treats problems as linear. At an extreme it measures the lethal dose 50 in animals – the dosage needed to kill half of those given it. At the other end, they make attempts to determine the highest dose at which no adverse effect is seen. The level that gets described as “safe” is determined by starting with doses that do cause harm, reducing it until a level is reached where what is being measured is no longer observed, then dividing by an uncertainty factor to give the safety limit. The methods used mean that any hazard that appears only at lower doses cannot be detected.
E4 says harm should be measured across the range of doses that might be efficacious as well but it only ever treats harm as something which increases as the dose rises. Nowhere does it suggest that a smaller dose might do more damage than a larger one. As with all toxicology the approach taken is to raise the dose and stop where side effects become unacceptable. The lower dose is assumed to be safer throughout this process. The assumption that lower doses are harmless is embedded so deeply that when the guideline does describe adverse effects occurring more often at a lower dose, it calls the excess “spurious”. E4 may have been correct in that the study gave the same people increasing doses and let them drop out once they had an adverse event. That meant susceptible people were never exposed to high doses. It would make more sense to test different people with different doses and follow them all up for a reasonable period of time and that is what E4 recommends but it is rarely done. The design of current studies cannot show whether lower doses cause particular adverse reactions. Because the dosage question is approached from the point of ensuring optimal efficacy the dose comparison trials are small and short. A typical study to compare doses checks three or four dosages on groups of around fifty people for about twelve weeks. These groups are far too small for any measure of safety. The approach to toxicology is not well placed to detect low dose harm.
With regards to folic acid, when the MRC Vitamin Study reported in 1991, its own investigators noted that a trial with enough power to demonstrate efficacy usually lacks the power to settle safety, so that “a judgment on safety needs to be taken on wider grounds”. No such safety study has ever been done. On dosage they observed that a lower dose such as 0.36 milligrams was likely to be effective as well, though possibly less so, and that a very large trial would be needed to compare the two.
The cancer concerns were dismissed after a 2013 Lancet meta-analysis pooled results from trials which included doses of a fraction of a milligram per day up to forty times that amount. They then reported a single average. That is a legitimate answer to the question of how much cancer there was overall, but it cannot answer whether the effect differs by dose. E4 says that the differences between separate trials usually make such an approach “unsatisfactory”.
What the folic acid data actually show
The limited evidence on folic acid safety indicates that the harm may not be greater at the larger dose. This is true for both the cancer data and the fetal death data.
Firstly, the cancer trials studying exposure below one milligram a day show cancer incidence about 20 per cent higher than in the placebo groups. However, trials on doses higher than this show no excess. Secondly, the only large randomised trial of a modest dose of folic acid in women at ordinary risk, recruited while planning a pregnancy, reported no significant effect on any category of fetal death. That is accurate only when each category is examined separately. From the trial’s own table the total number of pregnancy losses was 374 among 2,793 pregnancies in the treatment group against 304 among 2,660 in the placebo group, significant at a p value of 0.028. That total includes every pregnancy loss after a positive pregnancy test. Counting only later losses still results in an excess but it is no longer statistically significant. The treatment group were given a multivitamin so the excess cannot be attributed to the 0.8 milligrams of folic acid alone. The only other trial to use a dosage below 1 mg, and the only one at that dose with an arm giving folic acid without other vitamins, was too small to see a safety signal and was stopped before reaching its target size because recruitment had slowed. The researchers pooled what little data they had to report a benefit but did not pool the same data when reporting pregnancy loss. When a Cochrane review pooled it in 2015 the miscarriage rate was 31 per cent higher on folic acid, on twenty five events in total, such that it was not statistically significant.
Three of the four randomised trials in women who had already had an affected pregnancy used 4 milligrams. The largest of them, the MRC trial, found that the excess of miscarriage was not statistically significant with a relative risk of 1.06 but a confidence interval from 0.79 to 1.43. It therefore could not exclude an effect of the size the Hungarian trial recorded. It could be that the size of the trial was the issue rather than that there were no excess pregnancy losses at the higher dose. In 1997 Judith Hall declared that it would be unethical to repeat the trials, so this remains the full extent of the data available.
| Daily dose | Cancer incidence | Pregnancy loss |
| Under 1 mg | About 20 per cent excess in pooled randomised controlled trials | Significant excess of total fetal losses in the 0.8 mg multivitamin trial, though folic acid cannot be isolated as the cause |
| Above 1 mg | No excess cancer incidence, pooled randomised trials | No excess of miscarriage, relative risk 1.06 (0.79 to 1.43) |
Both safety signals appear in evidence from doses below 1 milligram a day and neither appears at higher, medicinal dosing. The intended mandatory fortification will expose the population eating ordinary quantities of wheat to doses under 1 mg a day. A medicine is prescribed at a particular amount each day for a period of time. Fortification results in variable dosing each day and over a lifetime.
In 1997 Nicholas Wald, who had coordinated the MRC trial, reanalysed its miscarriage figures to see whether there was evidence that folic acid causes fetal loss. He concluded it did not and defended that conclusion by saying that the trial had used about ten times the dose proposed for the population, suggesting that evidence “adds further weight against the miscarriage hypothesis”. Remember, it was his same paper which had said six years earlier that comparing 4 milligrams with 0.36 milligrams would need a very large trial to ensure the right dose for efficacy. That was never done.
What happens in the body at higher doses
The effect of folic acid on blood folate stores at different doses has been measured. Women of reproductive age were given either 1.1 milligrams or 5 milligrams of folic acid daily for thirty weeks. The higher dose produced about twice the plasma and red cell total folate concentrations, despite a more than fourfold increase in the amount taken. Circulating levels of unmetabolised folic acid did not differ significantly between the two groups. The researchers suggested there may be mechanisms by which the body can adapt to high folic acid intakes and limit exposure to unmetabolised folic acid. If that is true, that the body handles it differently at different doses, that has huge implications for safety testing.
There are several interactions of folic acid in the body that would account for such an observation. The principle can be thought of as like a pipe that bursts and causes a flood. Such an incident is noticed and dealt with within hours. However, there are not the same systems to deal with a pipe that leaks insidiously and that can rot a joist over two years because it causes harm below the radar.
| Step | Threshold | What happens above that dose |
| Absorption from the gut | Carrier proteins saturate at pharmacological doses | The remainder crosses by passive diffusion, so the proportion absorbed falls |
| Conversion by DHFR (dihydrofolate reductase) | Capacity of the enzyme is exceeded above about 260 to 280 micrograms | Unconverted folic acid appears in blood and circulates |
| Reabsorption by the kidney | When natural folates are low, they are scavenged from the urine by specific receptors which folic acid blocks and which can be saturated at high doses | Folic acid spills into the urine above a certain dose; at least a quarter of a 4 mg dose is excreted unchanged |
At therapeutic doses, absorption from the gut, enzyme saturation and renal spill all limit what acts on other tissues. Fortification doses do not reach the thresholds for absorption or renal spill, but they do exceed the capacity of the enzyme. At the fortification dose the body therefore produces unmetabolised folic acid while neither of the mechanisms that shed it is yet in play. That is a low dose window, in which a modest amount is handled differently rather than a scaled down version of the effect seen with a medicinal dose.
Timing
Almost every trial in the folic acid safety literature was based on a daily tablet. The result is a daily peak that is then cleared. Folic acid in flour will be in bread, pasta, biscuits and sauces, arriving at breakfast, at lunch, at supper and at points in between. Exposure could be for a lifetime.
Intermittent large doses are cleared by the body but continuous low doses are not cleared in the same way. Naltrexone showed that the duration of occupancy of a receptor can determine the effect. Folic acid binds folate receptor alpha more tightly than the natural reduced folates do, so where it is present it competes for the receptor. Unmetabolised folic acid was found in 38 per cent of US adults aged sixty and over in the 2001 to 2002 national survey, after an average ten hour fast (routine fortification of flour with folic acid was introduced in the US in 1998). A later national survey covering all ages found it in more than 95 per cent of people, despite using a test that could only detect higher levels. These surveys were not picking up temporary peaks after a meal. Taking folic acid as a pill creates a single large peak which the body clears. Being exposed through fortification seems to leave unmetabolized folic acid in the blood continuously. The studies to show how much that matters have not been done for folic acid. Importantly, methotrexate, which is a drug used as an immune suppressant and as chemotherapy and is carried by the same transporters as folate, is absorbed more completely when the same total dose is given as repeated small amounts. Safety testing for repeated low dose exposure to folic acid in a healthy population simply has not been done.
Deciding the upper safety limit
The upper limit of 1 milligram per day for safety was decided on the basis of a single chosen endpoint, namely case reports of neurological deterioration in people with undiagnosed B12 deficiency. Other possible harms were reviewed but the evidence was judged too weak to include or unrelated to a particular dose. The upper limit therefore should not be considered to be a level below which safety has been shown. Different doses will result in different concerns. Worryingly, the only work on safety of fortification prior to the change to the regulations has been a calculation of how many people might exceed this hypothetical upper limit. That is not a good way of assessing the dangers.
Burden of proof
It is not for people with concerns about safety to have to prove that folic acid is definitely unsafe in all people. When governments decide to mandate adding a drug to a staple food, the onus is on them to have carried out proper safety testing beforehand. They did not.
From 13 December 2026 folic acid exposure will become universal in the United Kingdom. It also becomes far harder to study safety at that point because any potential placebo group would be exposed through their diet.
Pharmacology has understood for more than forty years that doses and their effects on the body are not linear. Safety testing needs to account for different negative impacts at different doses too.
