Written by Alexandra Chambers | 17^th May 2025 | https://darkmatters.press

Executive Summary

This report outlines the formal key concerns regarding scientific and regulatory misconduct, predominantly by the European Food Safety Authority (EFSA) in its evaluation of 3-nitrooxypropanol (3-NOP), commercially known as Bovaer®. Based on a comprehensive review of the European Food Safety Authority (EFSA) Journal 2021 and the subsequent Food Standards Agency, (FSA, 2023) safety assessment paper, this report presents evidence of significant toxicological harm that was either downplayed or reframed to support regulatory approval -despite clear concerns that should have triggered further scientific research and precautionary measures.

1.Bovaer Primary Additive

1.1 Propylene Glycol (See also Section 3.4 for a chemical comparison of Propylene Glycol and 1,3 Propanediol)

Propylene glycol constitutes approximately 35% of the Bovaer additive (per UK FSA specifications, 2023). This is relevant because:

• Propylene glycol has a known toxic profile in felines, where it is associated with Heinz body anaemia and oxidative damage (Christopher et al., 1989).

The use of propylene glycol in cattle feed introduces risk to non-target species via environmental exposure (e.g. cats consuming contaminated dairy, waste or organ meat products or derivatives).

The liver and kidneys are well-established sites for the accumulation of lipophilic substances and metabolically active residues, owing to their central roles in biotransformation and excretion. Lipophilic compounds, in particular, tend to persist in these organs due to their high perfusion, enzymatic activity, and fat content, making them primary targets for toxic accumulation (Vugmeyster et al., 2021). This is particularly important for felines, who are uniquely susceptible to oxidative damage- representing a serious gap in the current regulatory oversight.

According to toxicokinetic analysis, 1,3-propanediol is a measurable metabolite of 3-NOP, detected in caecum, colon, and excreted in urine as both free and conjugated forms in studies (EFSA, 2021, p. 8). This indicates systemic absorption and hepatic metabolism, including glucuronidation -an enzymatic pathway known to be limited in felines (Plapp et al., 2015). 

While 1,3-propanediol is not classified as a lipophilic compound -it has a low logP value and is highly water-soluble- it is still a metabolically active diol that is processed primarily by the liver (Plapp et al., 2015). This means that although it does not bioaccumulate in fatty tissue, it can transiently concentrate in the liver and kidneys, where alcohol dehydrogenase (ADH) activity governs its breakdown. In species with altered or deficient metabolic pathways, such as cats, even polar compounds like 1,3-propanediol may pose risks due to metabolic bottlenecks, accumulation of reactive intermediates, or prolonged exposure to low-level residues. 

1.2 Regulatory Gap in Cat Food Safety: Propylene Glycol Use in the UK and EU

In the United States, the Food and Drug Administration (FDA) explicitly prohibits the use of propylene glycol in cat food, citing its established link to Heinz body anaemia in felines. The restriction is codified under 21 CFR § 500.50, which states that propylene glycol is “not generally recognized as safe for use in cat food” (FDA, 2023a). In addition, the FDA maintains Import Alert #72-06, authorising the detention of imported cat food containing propylene glycol without physical examination (FDA, 2023b). These regulations reflect a clear precautionary stance based on known feline toxicity.

In contrast, no such prohibition currently exists in the UK or the European Union. Propylene glycol is permitted as a food and feed additive under Regulation (EU) No. 1333/2008, which has been retained in UK law post-Brexit (European Parliament and Council, 2008). However, the regulation does not specify any restriction regarding its use in cat food, nor does it require labelling specific to feline exclusion. As a result, unless voluntarily avoided by manufacturers, the compound may legally appear in cat food products in these jurisdictions -despite its known risks to feline health.

This regulatory disparity represents a significant and troubling oversight. The absence of clear prohibition within the UK and EU framework creates a silent exposure pathway for a compound already banned in another major regulatory system. The fact that the U.S. has taken action while the UK/EU has not -despite scientific consensus on its toxicity to cats -raises serious concerns about the consistency and robustness of feline safety standards across borders.

The lack of traceability, public disclosure, or reassessment on Bovaer trials reflects a wider breach of transparency, regulatory continuity and constitutes a lack of informed consent for the public; not only on their food products, but also their pet food products.

2. Multispecies Signs of Harm 

2.1 Tolerance Studies in Cows Revealed Toxicity, Then Were Misrepresented

Two tolerance studies in dairy cattle showed:

• Lethargy, reduced feed intake, and premature euthanasia at high doses.

• Statistically significant reductions in red blood cell count, haemoglobin, ovary size, and increased liver enzymes.

These effects are biologically significant and indicative of early systemic toxicity. Yet EFSA (2021) dismissed them as having “no clinical relevance,” based on short-term observations and narrow control ranges. No long-term reproductive follow-up was conducted in cows, and mechanisms of ovarian atrophy were not investigated.

EFSA’s own tolerance studies in dairy cows revealed clear signs of physiological distress and early toxicity at higher doses of 3-NOP: 

• Cows dosed at 5× and 10× commercial levels developed lethargy and reduced feed intake -with two animals euthanised prematurely due to welfare concerns. 

In a second, larger trial, cows receiving 220.4 mg/kg dry matter showed: 

• Reduced dry matter and water intake. 

• Lower red blood cell count, haemoglobin, and haematocrit. 

• Elevated liver enzymes (ALT, LDH) indicating hepatic stress. 

• Altered coagulation markers (fibrinogen, prothrombin time). 

• Significant reduction in ovarian size. 

These are not subtle effects. The changes spanned multiple organ systems -blood, liver, metabolism, and reproductive tissue- and were statistically significant. 

Yet EFSA dismissed them as having “no clinical relevance,” based on short-term observations and narrow interpretation of control ranges. No long-term reproductive follow-up was conducted in cows, and no mechanism of ovarian atrophy was investigated. 

The decision to overlook these signals reflects a deliberate narrowing of the safety lens -prioritising product approval over full biological risk assessment. 

This cross-species toxicology thread reveals a pattern of reproductive suppression, neurological impairment, and biochemical dysregulation: 

• In rats: irreversible sperm damage and infertility 

• In mice: systemic toxicity, organ stress, and visible distress at high doses 

• In dogs: neurological collapse and seizure onset, with histological damage 

These signals are not isolated -they’re consistent across mammals. Instead of triggering regulatory pause or further testing, EFSA (2021) defined NOAELs within margin-of-error ranges of visible harm. 

2.2 Doses That Harm: Understanding Toxicology

 Many people reading toxicology data might assume that effects seen in animal studies only occur at very high doses. The actual evidence around Bovaer (3-NOP) and its primary metabolite NOPA tells a far more troubling story. 

2.2.1 What Were the Doses? 

Across multiple animal studies -rats, mice, and dogs- the adverse effects appeared at doses ranging from 100 to 700 mg per kilogram of body weight per day. This is a standard range for long-term toxicology testing, not an exaggerated stress test. 

To translate that: 

• For a 10 kg dog or child, 100 mg/kg equals just 1 gram per day. 

• For a 70 kg adult, 500 mg/kg equals 35 grams per day. 

 These are well within the normal boundaries of pre-market screening. 

2.2.2 Reported Effects at These Standard Doses

• In rats, 500 mg/kg/day caused irreversible infertility -testes damage, sperm depletion, and complete failure of pregnancy in females. 

• In mice, 700 mg/kg/day caused neurological symptoms -abnormal gait, closed eyes, slowed breathing, and a drop in food intake, along with thymus shrinkage and organ stress. 

• In dogs, 300 mg/kg/day caused tremors and epileptic-like seizures, requiring one dog to be euthanised after only 3 weeks of exposure. 

These aren’t speculative endpoints -they are documented in EFSA’s (2021) own review. 

2.2.3 The Real Problem? How Close the ‘Safe’ Levels Are to the Harm 

EFSA (2021) defined “safe” levels -called NOAELs (No Observed Adverse Effect Levels)-right on the edge of observable harm: 

• 100 mg/kg/day for male rats, despite seeing early signs of sperm disruption. 

• 300 mg/kg/day for dogs, even after tremors, seizures, and neurological decline. 

This means the safety margins are not conservative. They are set at the very border of toxicity -something that would never pass scrutiny in pharmaceuticals or food-grade chemicals. 

2.2.4 Contradiction Between Observed Toxicity and Approved Dose Level

One of the most alarming findings in this investigation is the contradiction between the results of EFSA’s own cattle tolerance studies and the dose later approved by the UK’s Food Standards Agency. In the EFSA 2021 evaluation, dairy cows were administered 3-NOP at a level of 220.4 mg/kg dry matter (DM) -only slightly above the FSA’s later approved limit of 200 mg/kg DM (EFSA, 2021, p. 15). At this level, significant haematological changes were observed, including reductions in haematocrit, haemoglobin concentration, and erythrocyte count. These effects are consistent with impaired oxygen transport and raised concerns about potential methemoglobinemia, which was also reported in other species exposed to high levels of 3-NOP or its metabolites (EFSA, 2021, p. 28–29).

Despite these toxic effects being clearly documented, for their safety assessment the FSA (2023) approved a safe dose of 200 mg/kg DM -a level only 20 mg/kg lower than the dose at which toxicity was observed. No additional studies were conducted to verify that this margin was biologically safe or justifiable. The approval instead relied on a declared “margin of tolerance of 2”, without reassessing the toxic threshold or providing evidence of safety at the upper limit (FSA, 2023, p. 9).

This decision places the accepted exposure level perilously close to the known threshold of harm, violating core principles of toxicological risk management. The failure to uphold a meaningful safety margin -particularly when physiological distress in test animals was recorded- represents a profound breach of both scientific integrity and animal welfare standards.

2.3 Inhalation Exposure in Animals -An Overlooked Toxicological Route

Despite acknowledging that 3-NOP is an eye and skin irritant and may be harmful by inhalation to humans, EFSA conducted no evaluation of inhalation exposure in animals. This is a critical omission given that 3-NOP is administered in powdered feed, with a reported dusting potential up to 390 mg/m³ -a level high enough to raise serious concern for confined animals routinely inhaling feed-level dust.

Unlike farm workers, dairy cows inhale at point-blank range, nose-deep in troughs during feeding. This constant, close-range exposure means that inhaled particles bypass first-pass metabolism, potentially entering systemic circulation without the detoxification step of ruminal degradation -a process EFSA repeatedly relies on to justify the additive’s safety when ingested.

No inhalation toxicity studies were conducted in ruminants. Instead, EFSA relied solely on a rat inhalation study that showed morphological changes in nasal tissue and methaemoglobin formation at both tested doses (1 and 5 mg/L). These adverse findings were dismissed as “non-severe,” but they raise a far more serious concern when considered in the context of non-consenting, continuously exposed animals, including:

• Youngstock in proximity to treated cows.

• Calves, with developing respiratory systems.

• Non-target species exposed via barn air or cross-contamination.

The lack of investigation into respiratory effects in livestock -and the assumption that feed-based ingestion safety applies to airborne exposure- constitutes a major regulatory gap and a potential ethical breach.

2.4 Neglected Exposure Pathways: Eye and Mucosal Irritation in Treated Animals

The EFSA (2021) and FSA (2023) reports confirm that 3-NOP is a known eye irritant based on OECD TG 437 studies. They also acknowledge that the substance is a fine particulate powder with high dusting potential (up to 390 mg/m³). Yet, the evaluation entirely omits the obvious risk to the animals themselves -particularly their eyes, nasal passages, and mucous membranes, which are directly and repeatedly exposed during feed intake.

Unlike human users -who are presumed to wear protective equipment- the animals are:

• Eating with their heads immersed in feed troughs, where dust is most concentrated.

• Snorting and inhaling fine particles at point-blank range.

• Exposing their eyes to unshielded, daily contact with an acknowledged irritant.

This is a fundamental failure in toxicological logic; the very livestock for whom this additive is authorised are receiving more intense and chronic exposure -yet no data is provided on:

• Corneal or conjunctival inflammation in cows.

• Nasal epithelium damage in ruminants.

• Respiratory distress, coughing, or mucous membrane effects.

There were no histological eye or nasal exams conducted in any livestock species. No visual health endpoints were tracked. No studies have tested whether long-term exposure damages ocular tissue, particularly in enclosed barns with poor airflow and shared dust exposure across multiple animals.

In short: this additive that irritates human eyes and nasal passages in small controlled doses, is being fed -without shielding- to animals who eat face-first off the floor, with zero assessment of their ocular health. This is careless and ethically indefensible in terms of animal welfare.

2.5 Reproductive Findings

One of the most significant concerns acknowledged in the UK Food Standards Agency (FSA) 2023 evaluation of 3-NOP was the decision by the Committee on Toxicity of Chemicals in Food (COT) to apply an unusually high uncertainty factor of 300 -triple the standard safety margin of 100- in determining the revised acceptable daily intake (ADI). This adjustment was made due to the “severity of effects on the male reproductive system and the steepness of the dose-response relationship” (FSA, 2023, p.9), following evidence from chronic and short-term toxicity studies in rats that showed marked reductions in testicular weight at relatively low doses. Specifically, the point of departure was based on a BMDL₅ (benchmark dose lower confidence limit) of 95.6 mg/kg bw/day for testicular weight reduction, with a proposed ADI of 0.3 mg/kg bw/day providing a margin of exposure of only 167 to the NOAEL for mesenchymal cell hyperplasia observed in the carcinogenicity study. These findings underscore that even subclinical exposures may carry reproductive risks. The steep dose-response curve observed implies that once effects begin, harm may escalate rapidly, particularly in sensitive populations -a signal typically reserved for high-risk compounds. This finding alone warrants serious precautionary action and further independent investigation.

2.6 Absence of Cattle Fertility Studies Despite Intended Use

The EFSA Journal (2021) confirms that no fertility or reproductive safety studies were conducted in cattle, despite the product Bovaer® 10 being explicitly authorised for “ruminants for milk production and reproduction.” While reproductive toxicity was assessed in rodents (via multigenerational and prenatal development studies), the FEEDAP Panel extrapolated safety conclusions to dairy cows without direct testing of reproductive endpoints -such as oestrus cycling, conception rate, gestational health, or calving success. The only tolerance studies in cows (conducted over 56 and 90 days) focused on feed intake and milk yield, not reproductive integrity. This omission is particularly concerning given that 3-NOP metabolites (notably NOPA) have been linked to testicular accumulation, hormonal disruption, and genotoxicity in rodent studies. Without cattle-specific fertility data, the safety of this additive for breeding animals -and downstream implications for foetal development and human exposure- remains insufficiently established.

There is no direct evidence that Bovaer is safe for fertility in cows, despite being authorised for milk production and reproduction. The only observed reproductive signal -shrinkage of ovaries- was seen but dismissed (EFSA, 2021). Regulators relied on rat data and indirect metabolic assumptions rather than targeted studies in cattle.

2.7 Discrepancies in Stability and Exposure Estimates

Significant inconsistencies were identified between the applicant’s claims and EFSA’s findings regarding the stability of 3-NOP in feed. While the applicant initially stated that only 10% of the active compound would be lost during pelleting, EFSA’s Animal Feed and Feed Additives Joint Expert Group (AFFAJEG) estimated the actual loss at 25.9% after three months (EFSA, 2023). The applicant later corrected their data to reflect a 17% loss, but EFSA still deemed the discrepancy noteworthy and requested further clarification. This raises serious concerns about the accuracy of exposure assumptions used in safety assessments. If feed manufacturers are instructed to compensate for this degradation by adding 10% more 3-NOP, as advised, inconsistent implementation could result in variable dosing—potentially exposing animals to higher-than-authorised concentrations. No robust mechanism for ensuring uniform compensation was presented. This undermines the reliability of the risk assessment and calls into question the validity of the established NOAEL (No Observed Adverse Effect Level), particularly given the already-documented toxicological effects at higher doses. The lack of clarity around degradation pathways also further complicates risk assessment for both target and non-target species.

3.0           Metabolites

3.1 Accumulation of Metabolites in the Intestines and Correlation with Tumour Findings

In the EFSA 2021 assessment, it was confirmed that the small intestine is a primary site for the metabolism of 3-nitrooxypropanol (3-NOP), with significant levels of the metabolites 3-hydroxypropionic acid (3-HPA) and 3-NOP glucuronide identified in this region following administration. This suggests a localised metabolic load in intestinal tissues. Additionally, several minor but structurally uncharacterised metabolites were found within the small intestine, adding uncertainty to the toxicological impact of long-term exposure.

Critically, a 2-year carcinogenicity study conducted in Wistar rats revealed an increased incidence of benign gastrointestinal mesenchymal tumours and mesenchymal cell hyperplasia in the small intestines of females treated with 3-NOP at 50, 100, and 300 mg/kg body weight per day. Although not deemed statistically significant, these tumours were rare findings and noted by EFSA as not likely to be by chance.

Notably, a separate ADME study using radiolabelled (3-14C)-3-NOP in lactating goats revealed that 14.87% of the total radioactive residues accumulated in the intestinal tract content, with only 8.99% detected in edible tissues and the rest assumed to be exhaled as CO₂ or bound to milk fractions (EFSA, 2021). The significant retention of metabolites in the intestinal tract -despite systemic clearance- suggests a localised exposure risk that may contribute to intestinal cellular stress or proliferation, consistent with the site-specific tumour emergence in the small intestines. This mechanistic plausibility strengthens the case for a re-examination of gastrointestinal carcinogenicity risk, especially in ruminants chronically exposed via oral ingestion.

The co-localisation of metabolic activity and tumour formation in the same organ system raises serious concerns regarding localised toxicity, chronic irritation, or genotoxic influence. It is particularly noteworthy that some of the metabolites present in the small intestine were not identified, precluding full assessment of their safety profile.

3.2 Evidence of Byproduct Accumulation

EFSA’s residue studies (2021, pp. 12–13) confirm that while 3-NOP itself is absent from milk and tissues, its by-product NOPA is consistently detected in both. Milk samples from cows dosed with Bovaer showed NOPA concentrations up to 3.66 µg/kg, with multiple samples above the threshold of quantification. Despite this, the panel dismissed further concern -partly by classifying 1,3-propanediol and related breakdown compounds as “non-toxic” due to their endogenous appearance. 

However, this logic collapses under scrutiny. 1,3-propanediol is not inert -it is a precursor to aldehyde intermediates (e.g. 3-HPA) and potentially reactive epoxides like 1,3-propylene oxide (oxetane). These were not tested for in milk or tissues. Nor was any secondary inhalation exposure considered from farm workers, children, or animals in shared airspace with metabolising livestock. 

In short, these findings show that metabolite residues do make their way into milk and potentially food chains -but the compounds that could be most harmful were not assessed at all. 

3.3.1 Milk Levels

While FSA regulators assert that:

“Concentrations of 3-NOP and its metabolites in milk and edible tissues are not expected to reach levels of concern,”

FSA, 2023 p.17

This EFSA (2021) and FSA (2023) assumption is contradicted by extensive toxicological evidence submitted within the same application. Multiple studies confirmed systemic distribution and tissue retention of 3-NOP metabolites – especially 3-hydroxypropionic acid (3-HPA) and 3-nitrooxypropionic acid (NOPA)- across critical organs including the liver, kidneys, adrenal glands, testes, and epididymis. In Wistar rats, 3-HPA accounted for up to 84% of total radiolabelled residues in testicular tissue just one-hour post-dose. Parallel studies in goats and cows demonstrated NOPA residues in plasma at levels up to 104 µg/kg, yet regulators dismissed concern on the basis of detection thresholds in milk, rather than accounting for long-term accumulation or inter-individual sensitivity. The FSA’s statement rests on limited quantification and an assumption of benign metabolism to propanediol or CO₂, without addressing the biological activity of transient intermediates like NOPA and 3-HPA -both of which underwent genotoxicity and testicular toxicity testing in multiple species, doses, and delivery routes. The assertion that there is no cause for concern therefore relies more on absence of visible crisis than presence of robust safety. Despite the scientific findings, regulators have relied on assumptions of metabolic breakdown and “non-detectable” levels in milk, while the full toxicological profile suggests potential systemic bioaccumulation, reproductive disruption, and genotoxic risk. The minimisation of these findings constitutes a failure of precaution -particularly when 3-NOP is proposed for use in dairy animals producing milk for human consumption.

3.3.2 Unresolved Safety Concern: NOPA Metabolites in Milk and Genotoxic Risk

Although EFSA concluded that 3-NOP residues in milk are not a cause for concern, the question of whether its metabolites -especially NOPA- are still present in milk remains unresolved. While the 2021 EFSA report quantified NOPA exposure using a worst-case concentration of 3.66 µg/L, it did not confirm whether all biologically active NOPA derivatives or breakdown products had been adequately identified or measured. This is critical, because NOPA has been flagged for potential genotoxicity and was subjected to extensive in vitro and in vivo mutagenicity assays. The EFSA 2021 report itself acknowledges that “the genotoxicity of 3-NOP cannot be ruled out” and that NOPA is the main metabolite responsible for the observed testicular toxicity in rats. Yet no long-term studies have investigated whether NOPA derivatives accumulate in milk or interact with other dietary components in a way that could elevate human risk -particularly in infants, toddlers, and other high-consumption groups. Without rigorous metabolic tracing or high-resolution milk metabolomics, the assumption that NOPA poses no risk to the consumer remains scientifically unsubstantiated.

Table 3 (EFSA, 2021 p.27) shows that infants and toddlers had the highest exposure per body weight, reaching 0.45–0.47 μg/kg bw/day, despite NOPA’s genotoxic potential not being scientifically ruled out. This exposure estimate is based on the highest milk concentration detected (3.66 µg/L) but assumes:

• Zero NOPA in other foods, and

• No accumulation or synergistic effects in sensitive or vulnerable populations.

Furthermore, EFSA (2021) used a health-based guidance value (HBGV) of 1 mg/kg bw/day, applying only an uncertainty factor of 100 -far lower than the factor of 300 later recommended by the UK’s COT for reproductive endpoints.

Given that:

• NOPA was found to accumulate in reproductive tissues in rats.

• Its genotoxicity is plausible.

• And infants are consuming proportionally more dairy

This regulatory conclusion lacks the precautionary rigor required for early-life exposure risk. EFSA’s reassurance that “no residue could be found in animal tissues” does not account for chronic, low-level intake during vulnerable developmental windows.

3.4 Confirmed Metabolite in Ruminants: 1,3-Propanediol

EFSA (2021) and AFFAJEG (FSA, 2023) confirm that 3-NOP is rapidly metabolised in the rumen into 1,3-propanediol, a compound that:

• Is a diol that is structurally and functionally similar to propylene glycol. In fact, they only differ in the position of a single carbon atom (NCBI, 2024a; NCBI 2024b).

• Is known to affect redox metabolism and may compromise feline health if ingested due to its structural similarity to propylene glycol.

• Has not been evaluated for downstream risk to non-target species.

The EFSA (2021) and FSA (2023) papers concluded that 1,3-propanediol would not be of concern in the target species, but no science backed safety margin was evaluated for bystander species –such as felines.

Although the EFSA’s 2021 assessment of 3-NOP indicated rapid metabolism and excretion in ruminants, the limited data on tissue residues, particularly in kidneys – a common ingredient in feline diets- necessitates caution. The potential for residual metabolites in organ meats underscores the need for comprehensive residue studies to ensure the safety of pet foods derived from animals supplemented with 3-NOP. 

3.5 Presence of Unidentified Metabolites

EFSA’s data confirm that 3-HPA is a primary metabolite of 3-NOP, present at higher concentrations than the parent compound in the liver, intestines, reproductive tissues, and bone marrow (EFSA, 2021, p.10). EFSA also explicitly acknowledged that not all metabolites of 3-NOP could be structurally characterised, particularly those formed in the rumen and liver (EFSA, 2021, p. 11). Despite this, no further investigation or toxicological profiling was required. The presence of unidentified breakdown products introduces a significant layer of uncertainty, as such compounds may still possess biological activity, toxicity, or bio accumulative properties. This raises serious safety concerns, particularly as the primary excretion route is via expired air, not faeces, making environmental and inhalation exposure likely. Tissue accumulation doubled with repeated dosing, confirming systemic retention of metabolic by-productsProceeding with commercial deployment without resolving these unknowns represents a serious breach of precautionary standards.

3.6 Inhalation Risks Documented and Dismissed

Acute inhalation studies in rats revealed:

• Methemoglobinemia at both 1 and 5 mg/L concentrations.

• Histological changes in the nasal cavity and altered blood oxygen transport.

Despite this, EFSA (2021) concluded that inhalation posed “no toxicological relevance” and failed to require follow-up studies for occupational or farm settings, even with its high dusting potential (390 mg/m³) and volatile metabolites. Acute inhalation studies in rats demonstrated that 3-NOP is not benign when airborne. Exposed animals developed methemoglobinemia, a condition where red blood cells lose their ability to carry oxygen efficiently. Histopathology showed nasal cavity irritation and tissue changes, and the substance was formally classified under GHS Class 5 inhalation toxicity. 

EFSA (2021) concluded that inhalation posed no toxicological relevance, and no follow-up studies were required for occupational safety, farm environments, or downstream inhalation by animals or humans. No modelling was done to account for real-world farm conditions -especially enclosed barns where metabolite accumulation in the air is likely. This is a serious omission for a compound known to produce volatile by-products like 1,3-propylene oxide (oxetane) and 3-hydroxypropionaldehyde (3-HPA). 

3.7 Environmental Release of Volatile Metabolites via Exhalation

The EFSA’s 2021 assessment of 3-NOP confirms that the additive is rapidly broken down in the rumen, producing a range of metabolites -including volatile compounds such as oxetane. While urinary excretion is the primary route, the Panel explicitly acknowledges that volatile byproducts may also be exhaled by animals into the surrounding environment. Despite this, no modelling or air concentration studies were conducted to assess the exposure risk in enclosed farm environments where livestock, workers, and bystander species -such as domestic animals- share the same airspace.

Oxetane, in particular, is a cyclic ether with known alkylating properties, capable of interacting with biological tissues upon inhalation. The EFSA Panel’s assumption that it is unlikely to persist, and thus not significant, is not supported by data. No toxicological reference value or environmental fate analysis was established, nor were atmospheric interactions or chronic inhalation effects evaluated.

This raises a fundamental breach of regulatory principle:

We must not base food safety -or environmental safety- on assumption.

To classify 3-NOP as environmentally beneficial while failing to assess the environmental release and toxicological relevance of volatile breakdown products is both scientifically negligent and misleading to the public.

4.0       Genotoxicity

4.1  Regulatory Precedent: Oxetane Derivatives and Genotoxicity

Evidence from the Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS, 2010) demonstrates that oxetane derivatives are not universally benign. In a 2010 public risk assessment, NICNAS reported that a specific oxetane compound -3,3′-[oxybis(methylene)]bis[3-ethyl]- exhibited positive genotoxicity in an in vitro mammalian chromosome aberration assay. This outcome directly challenges EFSA’s assumption that oxetane, a volatile metabolite of 3-NOP, poses no risk when exhaled into agricultural environments. Notably, EFSA (2021) did not conduct or request any genotoxicity testing specific to oxetane, nor did it account for the compound’s volatility and potential inhalation by co-housed animals or humans. The NICNAS findings support the conclusion that case-by-case evaluation of oxetanes is essential and that EFSA’s failure to assess this pathway represents a significant breach of the precautionary principle.

4.1.2 Core Sequence of Concern: 

• Bovaer (3-NOP) is administered to livestock to reduce methane emissions. 

• Inside the body, 3-NOP metabolises into 3-hydroxypropionaldehyde (3-HPA). 

• 3-HPA is an aldehyde: reactive, potentially toxic, and capable of converting into acrolein under oxidative or thermal conditions. 

Acrolein is a well-known volatile toxin that causes: 

• Respiratory inflammation and damage. 

• Mucosal erosion. 

• Immune suppression. 

• Neurological distress in sensitive species. 

Key Concerns: 

Volatility: Acrolein can be exhaled or released environmentally. 

Shared airspaces: This exposes other animals (e.g., cats, birds) to secondary inhalation. 

Symptoms may resemble respiratory infection, creating a misdiagnosis risk – potentially mistaking toxicological reactions for viral conditions such as bird flu. 

4.1.3 3-HPA is a Primary Metabolite: 

“In the small intestine… the most representative [compound] being 3-HPA.” 

“In the liver… 3-HPA was detected at higher levels than the parent compound.” 

“In the epididymides and testes, 3-HPA was the major compound.” 

This reaffirms: 3-HPA is not incidental. It is a major metabolite -in key tissues like the liver, reproductive system, and intestines. 

This directly supports a link to aldehyde toxicity and potential acrolein formation. 

 4.1.4 Mechanistic Plausibility: 

• 3-HPA volatility is limited, but conversion to acrolein increases with environmental exposure (heat, UV, air). 

• Birds, cats, and small mammals in proximity to Bovaer-treated livestock may inhale trace levels of acrolein from exhaled air or barn environments. 

4.1.5 Regulatory Oversight Gaps: 

• No formal inhalation risk analysis of 3-HPA to acrolein conversion in farm settings.

• No veterinary safety data on chronic, low-dose exposure to acrolein from metabolic origin. 

• No multi-species risk assessment despite shared air and environment. 

“Excretion happens mainly in expired air as CO₂…” 

According to EFSA (2021) toxicokinetic data, 3-NOP and its metabolites were widely distributed across organ systems, with peak concentrations in liver, kidneys, and adrenals, and measurable radioactivity in lung, spleen, and pancreas (EFSA, 2021, p.9). Volatile metabolite exhalation accounted for up to 80% of dose elimination, raising concerns over airborne by-product exposure -particularly in enclosed or multi-species environments. 

4.2 Suppression of Reproductive Toxicity Data in Rodent Studies

Multiple rodent studies reported:

• Irreversible spermatogenic arrest, tubular degeneration, and testicular atrophy in males at 300–500 mg/kg/day.

• Complete failure of implantation and pregnancy in females at 500 mg/kg/day.

• Reduced ovary size and failed corpora lutea development.

Despite these outcomes, EFSA (2021) concluded that the ‘No Observed Adverse Effect Level’ (NOAEL) was 100 mg/kg/day and declared the compound safe for livestock consumption. However, significant reproductive toxicity was observed at 500 mg/kg bw/day, including irreversible spermatogenic arrest and complete failure of implantation and pregnancy. The narrow margin between the NOAEL and these severe effects, along with unresolved questions about 3-NOP’s genotoxicity, raises concerns about the adequacy of the safety assessment and underscores the need for further research. 

4.3 Minimisation of Genotoxicity: Organisation for Economic Co-operation and Development (OECD) Standards and Testing

The European Food Safety Authority (EFSA, 2021) and Food Standards Agency (FSA, 2023) paper confirm that multiple in vitro studies identified statistically significant genotoxic effects of 3-NOP:

• Two in vitro micronucleus assays showed clear positive results (including in Chinese hamster V79 cells).

• A third yielded equivocal results.

• An in vivo study in rats (oral administration) also showed a statistically significant increase in micronuclei.

EFSA dismissed all genotoxicity concerns, citing:

• “Historical control ranges,”

• A Giemsa-based staining artefact claim.

EFSA (2021) and FSA (2023) ultimately dismissed the genotoxic findings based on a claim that the micronucleus signals were artefacts caused by Giemsa staining -an assertion made not by an independent reviewer, but by a “consultant directly contracted by the applicant themselves” (FSA, 2023, p.8). This is not a scientific assessment; it is a narrative accepted without independent replication or oversight.

Micronucleus assays in mammalian cells (TK6 and V79 lines) showed:

• Statistically significant increases in micronucleated cells under metabolic activation.

• One Chinese hamster study (EFSA, 2021 p.15) found a significant 79% of micronuclei contained chromosomal fragments, indicating clastogenic activity.

NOPA, a major metabolite of 3-NOP, induced dose-dependent gene mutations in multiple Salmonella strains, indicating genotoxicity (EFSA, 2021 p.17 Bacterial reverse mutation assay-Ames- test).

4.3.1 Mutagenicity of the NOPA Metabolite in Ames Test:

One of the most concerning findings in EFSA’s 2021 toxicological review was the mutagenicity of NOPA, the primary metabolite of 3-NOP, in bacterial reverse mutation assays. The additive induced substantial and dose-dependent gene mutations in Salmonella Typhimurium strains TA1535 and TA100, both with and without metabolic activation (EFSA, 2021, p. 15). This result, observed across a wide dose range (3 to 5,000 µg/plate), suggests that NOPA can cause base pair substitutions, a hallmark of genotoxic potential. These findings meet OECD criteria for a clearly positive result in Ames assays and were not adequately reconciled with the overall regulatory conclusion of non-genotoxicity.

In conclusion, NOPA induced gene mutations by base pair substitutions in the genome of strains TA1535 and TA100 in the presence and absence of S9 mix.” (EFSA, 2021, p. 15)

These findings meet OECD criteria for genotoxic concern. However, EFSA (2021) downplayed or excluded these results from their final risk narrative by citing methodological limitations (e.g., non-GLP protocols) without requiring confirmatory studies.

4.3.2 Limitations and Findings of the In Vivo Micronucleus Assay in Rats

In the 2021 EFSA assessment, an in vivo micronucleus test was performed using Fischer 344 Big Blue® rats to evaluate the potential of NOPA (a metabolite of 3-NOP) to induce chromosomal damage in peripheral blood reticulocytes (EFSA, 2021, p. 17–18). While no statistically significant increase in micronucleated reticulocytes (MN-RETs) was observed, a significant decrease in reticulocyte percentage was reported in male rats at day 29 -an indicator of cytotoxicity. Despite this, the author’s concluded NOPA was negative for genotoxicity in this test. Notably, no concurrent positive control was included, undermining the assay’s sensitivity and interpretive strength. Instead, historical control data and manufacturer-supplied positive controls were used. These limitations, alongside prior positive genotoxicity signals in other assays (e.g. Ames and Chinese hamster V79 cells), suggest that this result should not be viewed as definitive evidence of safety, but rather part of a mixed and contradictory dataset.

4.3.3 Use of Outdated OECD Test Guidelines in EFSA’s 2021 Assessment and Failure to Reassess Under Updated Standards (FSA 2023)

EFSA’s 2021 safety evaluation of Bovaer (EFSA, 2021) appears to rely on toxicological studies conducted under OECD Test Guidelines that have since been revised significantly to reflect advancements in scientific understanding and methodology. Notably:

• OECD TG 403: The acute inhalation toxicity study was conducted following TG 403, which has been updated to recommend alternative methods such as TG 436 (Acute Toxic Class Method) and TG 433 (Fixed Concentration Procedure) that utilize fewer animals and provide more refined data on inhalation toxicity (OECD, 2009a; OECD, 2009b). 

• OECD TG 474 and TG 487: Genotoxicity assessments were based on TG 474 (Mammalian Erythrocyte Micronucleus Test) and TG 487 (In Vitro Mammalian Cell Micronucleus Test). TG 487, in particular, underwent revisions in 2023 to enhance clarity and precision in detecting clastogenic and genotoxic effects (OECD, 2023). 

The reliance on these earlier versions without incorporating the updated methodologies may have led to underestimation of potential risks associated with Bovaer, particularly concerning inhalation exposure and genotoxicity, particularly relevant for toxicological thresholds for volatile metabolites such as oxetane. Given the revisions in these guidelines, a re-evaluation of Bovaer using the current OECD TGs is warranted to ensure a comprehensive and accurate risk assessment. 

In EFSA’s 2023 re-evaluation of Bovaer (EFSA, 2023), no recommendation was made to update or repeat the key toxicological studies -despite clear revisions to the relevant OECD guidelines in the intervening years (OECD, 2009a; OECD 2009b; OECD, 2014; OECD, 2023; OECD, 2023b). This omission undermines the reliability of the risk assessment and highlights the need for a full re-evaluation under updated international safety standards.

According to OECD guidelines and global genotoxicity protocols, any indication of clastogenicity -regardless of study perfection- requires a validated, follow-up assay under standardised conditions. This is especially critical when: 

• The compound is metabolically activated (which mirrors actual use in livestock). 

• The results suggest chromosomal fragmentation. 

• The affected systems are mammalian or human-relevant. 

The EFSA (2021) and FSA (2023) have failed to meet that standard. 

4.3.4 Breach of the Precautionary Principle (UK Government Article 7, Regulation (EC) No 178/2002) 

The precautionary principle, enshrined in EU food law, mandates that when scientific uncertainty exists about potential risks to human or animal health, protective measures must be taken -even in the absence of conclusive evidence. In the case of 3-NOP and its metabolite NOPA, the EFSA panel acknowledged multiple ambiguous findings, including statistically significant clastogenic effects in in vitro micronucleus assays, unexplained metabolite pathways (e.g. oxetane-like intermediates), and multiple adverse histopathological effects in laboratory animals -yet concluded “no concern” for genotoxicity and systemic toxicity without requiring further targeted studies. 

This standard was not upheld:

• EFSA acknowledged positive genotoxic signals but dismissed them without consensus or transparency.

• Critical safety claims were based on industry-contracted consultants.

• The toxic profile of metabolites (e.g. 1,3-propanediol) is assumed safe for ruminants but never evaluated for domestic pets, despite the realistic pathway via milk and organs.

This failure to trigger precautionary oversight despite clear concerns -especially given the additive’s systemic exposure via food chains -constitutes a direct violation of the precautionary principle and undermines public trust in risk governance. 

4.4 Inhalation Exposure and a Neglected Genotoxic Pathway

EFSA’s own 2021 report acknowledged the presence of volatile breakdown products, including oxetane, following the ruminal metabolism of 3-NOP. Oxetane is a small, strained cyclic ether with known alkylating potential -a chemical class associated with DNA damage and genotoxicity. While a respiratory tolerance study was conducted in cows, this assessment focused on acute clinical signs and did not include any testing for genotoxic endpoints such as micronuclei, DNA strand breaks, or mutagenesis via inhalation exposure.

This is a critical omission.

EFSA’s conclusion that 3-NOP is not genotoxic relied on in vitro mammalian cell assays- two of which were clearly positive -and a single in vivo oral study in rats. At no point did the panel investigate whether inhaled metabolites, particularly oxetane, could be the underlying cause of the observed genotoxicity. The absence of any inhalation-focused genotoxicity studies, despite the confirmed exhalation of reactive volatile compounds, represents a major flaw in the risk assessment process.

This could represent the true mechanistic pathway of genotoxicity in exposed animals and humans -a pathway left completely unexamined.

A paper by Wright et al., 2010 explored oxetanes as a chemical motif and concluded that some oxetane derivatives possess measurable alkylating potential- particularly in the context of protein and DNA interactions -highlighting a need for case-by-case evaluation of their genotoxicity.

5.   Contradictory Conclusions and Strategic Narrative Framing

The EFSA (2021) and the FSA (2023) reports includes internal contradictions and language minimisation:

• Acknowledging that genotoxicity “cannot be ruled out” (EFSA 2021, p18) then later FSA 2023 stating “The Group concluded that 3-NOP is non-genotoxic in vivo. The Group concluded that the metabolite NOPA is non-genotoxic in vivo.” (FSA 2023 p8).

• “The AFFAJEG compared their exposure assessment results to those presented by the applicant and concluded that, based upon the LLOQ concentration of 5 µg/kg, the levels of NOPA residues in milk were low enough not to be cause for concern,” disregarding genotoxicity (EFSA, 2023 p.10).

• Dismissing statistically significant findings as within historical control ranges (EFSA, 2021; FSA, 2023).

This selective dismissal of adverse outcomes reveals a disturbing pattern: evidence was not absent -it was reframed.

The FSA 2023 panel have explicitly acknowledged in their assessment that the “environmental risk assessment submitted by DSM (the applicant) deviated significantly from standard regulatory guidance” (FSA, 2023, p.11). Despite this, the FSA proceeded to accept the applicant’s conclusions and supported the continued approval of the additive for use in the UK.

This raises serious concerns about the integrity of the regulatory process. The phrase “significant deviation” is not a minor caveat -it is an acknowledgment that the submission did not meet the basic standards of scientific scrutiny. In a functional system, such a deviation would prompt further data requests, external peer review, or even rejection of the application.

The fact that the FSA chose to proceed regardless suggests a worrying degree of regulatory leniency -particularly in relation to a novel additive with limited long-term data, known nitrate/nitrite and propanediol metabolites, and potential downstream effects on non-target species.

It is unacceptable for safety decisions to be based on incomplete or flawed assessments, and even more concerning when proof of harm is acknowledged and yet dismissed. This undermines public trust in the FSA’s capacity to act independently, transparently, and in the interest of public and environmental health.

The FSA conclusion: “An acceptable risk to the environment” (FSA, 2023 p.11).

6.   Conclusion 

The micronucleus assay results in EFSA Journal 2021 (pp. 15–16) should have triggered immediate, precautionary replication using a fully compliant GLP protocol. Instead, they were quietly dismissed. This undermines the integrity of regulatory oversight and exposes both livestock and bystander species -including humans- to an unquantified genotoxic risk. 

Bovaer is marketed as a climate-friendly solution intended to reduce methane in the atmosphere. Yet its metabolic by-products are expelled into the air- not as inert gases, but as potentially toxic aldehydes, including precursors to acrolein. 

This regulatory omission is not trivial. It means: 

• A feed additive with documented chromosome-breaking potential, in metabolised form, is now widely administered. 

• No human follow-up has been conducted on inhalation exposure, low-dose chronic exposure, or foetal sensitivity. 

• No independent peer-reviewed genotoxicity study has replicated or refuted the clastogenic findings. 

EFSA’s decision not to replicate the test has resulted in an unverified chemical being introduced into milk production chains, enclosed air environments, and multi-species ecosystems -despite evidence that its breakdown products may cause human and animal DNA damage under conditions that closely mimic reality. 

The effects on cattle themselves -who are the primary recipients of this additive -remain dangerously underexamined. Preliminary data points to possible reproductive disruption, impaired fertility, altered gut flora, and immune dysregulation, none of which have been studied beyond short-term, industry-sponsored trials. Additional animal welfare data on eyes and respiratory distress, given the context of the feed additive remain unexamined. The metabolite oxetane, though not detected in residue tests, may still form transiently in vivo and exert mutagenic effects at the cellular level. Over time, this could result in reduced calving success, metabolic disorders, shortened lifespan, and increased susceptibility to disease. In dairy herds already under immense physiological strain, the introduction of a compound with documented clastogenic properties is reckless -particularly when no long-term fertility or epigenetic studies have been published. The animals being sold as climate solutions may, in time, become the earliest casualties of a product designed and deployed without adequate biological caution.

If Bovaer continues to be deployed without transparent, long-term, cross-species safety testing, the consequences could be devastating. Primary metabolites have demonstrated clastogenic activity, meaning they can damage DNA, potentially leading to infertility, immunosuppression, or cancer over time. The compound’s intermediary metabolite, Oxetane, is particularly concerning -this unstable, four-membered ring is highly reactive and genotoxic, and its presence in metabolic pathways raises alarms for mutagenicity, DNA alkylation, and cross-species toxicity. In non-target species -pets, wildlife, or any creature exposed through feed, runoff, or residue- this substance could trigger neurological damage, hormonal disruption, and irreversible genomic instability, especially in small or genetically sensitive organisms.

For humans, especially those already vulnerable due to methylation defects, or immune compromise, chronic low-dose exposure through meat, milk, environmental leaching, or inhalation could result in accumulated DNA damage, carcinogenesis, or transgenerational epigenetic shifts. The assumed inert breakdown products -nitrate, nitrite, 1,3-propanediol, and oxetane intermediates-are not benign. They are biologically active, largely unstudied, and systemically dismissed in regulatory shortcuts. Bovaer is a high-risk chemical intervention and its long-term legacy could be widespread biological harm across species, ecosystems, and human DNA.

These Bovaer by-products could pose great risks to humans and animals, as well as local ecosystems and air quality- significantly more than the methane they were intended to replace. 

I respectfully submit this report as evidence of regulatory bias and scientific minimisation in EFSA’s approval of 3-NOP. I also request that the European Ombudsman and relevant oversight bodies:

• Open an inquiry into EFSA’s interpretation and framing of toxicological data.

• Require full, GLP-compliant reproductive toxicity and long-term inhalation studies in cattle.

• Reconsider the current authorisation of Bovaer® in livestock feed pending independent investigation.

• Provide clear transparency on all affected product supplies to enable informed choice for consumers.

I further request that all existing data related to 3-NOP be subjected to independent re-analysis by an impartial scientific body. Continued authorisation without such reassessment risks public trust, food chain integrity, and human and animal safety and welfare.

7.     Recommendations

It is also recommended to immediately review:

• All authorisations related to 3-NOP-containing additives.

• The safety of propylene glycol at 35% inclusion rate in ruminant feed, particularly for non-target species.

• Risk of 1,3-propanediol contamination in non-target species (especially felines).

• Breach of the precautionary principle and improper dismissal of statistically significant genotoxicity data.

A comprehensive full investigative report into Bovaer is currently being finalised and will be made publicly available in due course. This letter and its outcomes will also be included in the report.

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