By Alma Laney and Alison Bernstein
This post is the third in a series about transgenerational inheritance, epigenetics, and glyphosate that address questions raised by the publication of the paper, Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology.
Is the dose of glyphosate appropriate?
In any study on glyphosate, the dose must be comparable to actual exposure in order to provide useful results. For background on exposure, view SciMoms’ series on Risk vs Hazard.
The chosen dose was 25 mg/kg/day via intraperitoneal (i.p.) injection, which is an injection made through the peritoneum, which is a thin membrane lining the abdominal cavity. This exposure paradigm is different than dietary, inhalation or dermal exposure, which are the primary routes of potential human exposure, but agents administered via i.p. do go through the liver and this is a commonly used method. As with any experimental design choice, there are pros and cons to any delivery method. It is important to keep exposure route in mind when considering the results.
The authors write: “Twenty-five mg/kg for glyphosate is 0.4% of rat oral LD50 and 50% of the NOAEL and considering glyphosate rapid metabolism approximately twice the occupational exposure 3–5 mg/kg per daily exposure.”
LD50 is not relevant to this model of toxicity and only tell us about risk in cases where someone is exposed to a large amount of a chemical in a short amount of time. In other words, LD50s are relevant for accidents, murders or suicides. Reference dose (RfD) or Acceptable daily intake (ADI) is the relevant number when looking at chronic toxicity and is calculated from the No Observed Adverse Effect Level (NOAEL).
RfD (Reference Dose) or ADI (acceptable daily intake): an estimate of the daily exposure to humans that is likely to be without appreciable risk of deleterious effects throughout the entire lifetime.
In lay language, the reference dose is the amount a person could consume every day of their entire life and still be safe.
In the most current human health assessment from EPA, the NOAEL is 175 mg/kg/day and the RfD is 1 mg/kg/day. In the EU, EFSA based their RfD calculation on a NOAEL of 50 mg/kg/day and calculated an RfD (called ADI in the EU) of 0.5 mg/kg/day. The dose in this paper of 25 mg/kg/day is 25 times higher than the EPA RfD and 50 times higher than the EU ADI. This dose is 50% of the EFSA NOAEL and if the effect is real, this would suggest that the NOAEL is not actually a NOAEL. This is important if they are trying to do research to establish regulatory limits, but not relevant to understanding the effect of actual human exposures (see below for comparison to actual human exposure data).
Some may notice that the RfD in the new EPA draft human health assessment is higher than the old RfD for glyphosate of 0.1 mg/kg/day. If we use the older, lower RfD, the chosen dose is even more irrelevant at 250 times the EPA RfD. The new higher limit is based on additional studies done since the previous registration for glyphosate and excludes the previous study that showed an adverse effect in the F3 generation. This exclusion explained in the new draft assessment (See section 4.4.3 of the above-linked PDF for details).
In the three-generation study conducted in 1981 prior to the institution of the current Test Guidelines and Good Laboratory Practices, focal tubular dilation of the kidneys was observed in the offspring. This finding was judged to be spurious and unrelated to treatment since more extensive evaluations in subsequent reproduction studies conducted at much higher doses did not replicate the offspring effects.
For more detailed information about what “safety” means in a regulatory setting and what these metrics (LD50, NOAEL and Reference Dose) mean, read “Defining Safety: How Safe is Safe?” and “Glyphosate Vs. Caffeine: Acute and Chronic Toxicity Assessments Explained”.
Their quoted occupational exposure level is inconsistent with current data from the Agricultural Health Study. In the Farm Family Exposure Study data summary (part of the Agricultural Health Study), 60% of pesticide applicators in the study had detectable levels of glyphosate in their urine and the average urine level was 3.2 parts per billion (ppb). Average urine levels for spouses and children of applicators was less than 1 part per billion with only 4% and 12%, respectively, of each group having detectable exposures.
When exposure levels were estimated from these urine levels in this peer reviewed paper, it was found that the average exposure of 3.2 ppb for applicators corresponds to a dose of 0.001 mg/kg/day, or 0.1% of the newest EPA RfD and 0.2% of the EU ADI. Even the highest urine level of 223 ppb reported in the Farm Family Exposure Study corresponds to 0.004 mg/kg/day (0.4% of the EPA RfD) for the most highly exposed individual who didn’t take appropriate safety precautions. The dose in this paper of 25 mg/kg/day is 6,250 times higher than the highest measured exposure in the Farm Family Exposure Study and 25,000 times higher than the average measured exposure in this study.
Using even the highest estimates of exposures, pesticide applicators are exposed to levels of glyphosate that are a very small percentage of the safe limits. Consumers are exposed to much lower levels. Measures across other studies of agriculture and dietary exposures were consistent with these results. These estimates are far lower than the dose used in the paper and lower than their quoted estimate of 3-5 mg/kg daily exposure for occupational exposures.
Once we noticed these problems in their dose, we tracked their citations for this claim of a 3-5 mg/kg daily exposure and found citation errors (a problem that occurs at least one other time throughout the paper). Their citations for this exposure level are:
- A paper that looks at incidents of intentional self-poisoning that reports 4 cases of self-poisoning. In toxicological-speak, Intentional self-poisoning is acute dosing with very high doses (examples in the paper include: 85 g with 2-3 liters of beer, 18-35 g,1 Liter but no actual dose reported, and 72-91 g). Thus, this is an irrelevant citation for their occupational exposure number.
- An EFSA report that estimates various exposure scenarios as a % of AOEL (Acceptable operator exposure levels). Exposure levels here correspond to a daily dose of 0.1-0.66% of the ADI. If we take the highest dose (0.66% of ADI), this corresponds to 0.33 mg/kg/day. The dose used in this paper is 75 times higher than this highest estimated occupational exposure.
With this choice of dose and route, it is unclear what the authors are trying to model in this study. We can certainly do toxicology by increasing doses until we see an effect (this is important for finding limits and how high we can safely go), but this type of toxicology does not model what might be actually happening in people at relevant exposure levels. Thus the reporting of this paper as relevant to human exposures, even the highest of occupational exposures, is not justified.
Above, we mentioned that the citations supporting their claim of relevance to occupational exposure did not actually support that claim. While we did not examine the accuracy of every citation in the paper (there are 100 of them after all), the citations were incorrect in the only two places where we went to check citations.
The second place is the results with this sentence: “Direct exposure studies to glyphosate have been shown to induce behavioral abnormalities in the exposed F0 generation”. The authors then cite 4 papers that are not about glyphosate. These 4 papers include: a paper about vinclozoline, a review about transgenerational effects of endocrine disruptors that does not include the word glyphosate, a paper on atrazine in rats and a paper on atrazine in mice. These reference errors are sloppy at best but raise red flags and indicate that this paper requires closer scrutiny.
View the other parts of our series on transgenerational epigenetic inheritance:
- Transgenerational Epigenetic Inheritance and Glyphosate: Do we need to think of the grandchildren? (Introduction)
- Part 1: What is transgenerational inheritance?
- Part 2: Guidelines for studying epigenetic inheritance
- Part 3: Is the dose of glyphosate appropriate? (this post)
- Part 4: The problem of founder effects
- Part 5: Statistics
- Part 6: Methylation analysis