Reproductive & developmental toxicity of quaternary ammonium compounds

Abstract

Quaternary ammonium compounds are a class of chemicals commonly used as disinfectants in household and healthcare settings. Their usage has significantly increased in recent years due to the COVID-19 pandemic. In addition, quaternary ammonium compounds have replaced the recently banned disinfectants triclosan and triclocarban in consumer products. Quaternary ammonium compounds are found in daily antimicrobial and personal care products such as household disinfectants, mouthwash, and hair care products. Due to the pervasiveness of quaternary ammonium compounds in daily use products, humans are constantly exposed. However, little is known about the health effects of everyday quaternary ammonium compound exposure, particularly effects on human reproduction and development. Studies that investigate the harmful effects of quaternary ammonium compounds on reproduction are largely limited to high-dose studies, which may not be predictive of low-dose, daily exposure, especially as quaternary ammonium compounds may be endocrine-disrupting chemicals. This review analyzes recent studies on quaternary ammonium compound effects on reproductive health, identifies knowledge gaps, and recommends future directions in quaternary ammonium compound–related research.

Summary Sentence 

Quaternary ammonium compounds, a class of disinfecting compounds that have skyrocketed in usage during the COVID-19 pandemic, are emerging as reproductive and developmental toxicants.

Introduction

Quaternary ammonium compounds (QACs) are a class of synthetic chemicals found in common disinfectant and antiseptic cleaning items, as well as drug, personal care, and hygiene products [1]. Within the United States, QAC products with the intent of disinfecting are considered pesticides and regulated under the Environmental Protection Agency (EPA); however, QAC products that do not have the purpose of disinfecting are instead managed by the Food and Drug Administration [2]. In this study, we review QACs used by consumers for disinfection and in personal care products.

Humans are continually exposed to QAC-containing products during everyday use in household, medical, and public settings. They are widely used in disinfecting products due to their ability to destroy bacteria and viruses on surfaces. They are also found in many personal care products, specifically hair care. Examples of commonly used QAC-containing products include sprays, wipes, eye drops, mouthwash, and shampoos [3, 4]. Although QACs were developed approximately one century ago, they have recently gained market share as other antimicrobials, such as triclosan and triclocarban, have been deemed unsafe [4, 5]. In addition, QACs grew in popularity throughout the COVID-19 pandemic [6]. They are an active ingredient in over 200 cleaning products on the EPA’s N list, a list of disinfectants that are effective in protecting against the SARS-CoV-2 virus [7]. The sales of Lysol wipes, a standard QAC-containing disinfectant product, increased by 50% in the spring of 2020 alone [8]. Following the COVID-19 pandemic, the global market for QACs is valued at 1.08 billion USD and is expected to continue to rise [9]. The EPA classifies QAC mixtures, alkyldimethylbenzylammonium chloride (ADBAC), and didecyldimethylammonium chloride (DDAC) as “acutely toxic” via ingestion, inhalation, and dermal contact, with oral and inhalation exposure, as well as irritation and corrosivity to the skin from dermal exposure as additional concerns [4]. With chronic use of QACs, the most well documented effects include skin and/or respiratory-related illnesses such as asthma exacerbation, chronic obstructive pulmonary disease (COPD), anaphylaxis, dermatitis, nausea, vomiting, and eye irritation [4].

Structurally, these compounds have a cationic central nitrogen atom with four hydrocarbon substituents (Table 1). Quaternary ammonium compounds are positively charged, allowing them to efficiently attack the negatively charged surfaces of most bacteria. These chemicals degrade the membrane, leading to cytoplasm leakage and ultimately, inactivation [6]. They also bind to anionic surfaces such as hair and skin and are used in personal care products to impart softness and anti-static properties [10].

Table 1.

Common quaternary ammonium compounds

Commercial name	Purpose	Chemical structure
Alkyldimethylbenzyl ammonium chloride (ADBAC)/benzalkonium chloride (BAC) Note: ADBAC and BAC have the same basic parent structure, but the proportions of different side chains in the mixtures vary.	Surfactant, cleaning agent, antimicrobial agent
Didecyldimethyl ammonium chloride (DDAC)	Fungicide, antiseptic, disinfectant
Cetylpyridinium chloride (CPC)	Antiseptic
Alkyltrimethylammonium chloride (ATMAC)	Disinfectant, fabric softener, hair conditioner, emulsifying agent
Tetradecyl-dimethyl-benzyl-ammonium fluoride (TDBAF)	Spermicide
Ethyl(dimethyl) (tetradecyl) ammonium ethyl sulfate	Surfactant, emulsifier

With rising consumption rates, it is essential to understand the effects of QACs on the human body, especially from chronic low-dose exposure. While this class of compounds is known to have a risk of associated allergic reactions and respiratory complications with regular use, there is now emerging concern for these products threatening human reproductive and developmental health [2, 11, 12]. In this review, we break down the known information on the impacts of QACs on reproductive health in human-translational models. We analyze the existing literature on the effects of QACs on male and female reproductive health and embryo development, discuss existing gaps in current literature, and make suggestions for future investigations to provide more information on the health risks of QACs.

Methods

We identified studies in the literature with the goal of including all recent published studies on the reproductive and developmental toxicity of QACs used in consumer products. Quaternary ammonium compounds are also used as agricultural chemicals, such as the herbicide paraquat, and the plant growth regulator chlormequat; however, agricultural chemicals are not direct additives to consumer products and are beyond the scope of this review. Published studies from 2000 to June 2024 were included in this review to capture recent literature since the discovery that QACs used in disinfection of animal facilities were interfering with rodent reproduction [13]. The sources gathered for this assessment include studies that investigated QACs as reproductive and developmental toxicants in adulthood or childhood, as well as during pregnancy and from in utero exposure. We included in vitro studies that directly investigated endocrine mechanisms of action for QACs. Sources were identified using PubMed, Google Scholar, and Web of Science with keywords such as: “quaternary ammonium compounds reproductive toxicity,” “quaternary ammonium compounds developmental effects,” and “quaternary ammonium compounds neonatal” and searches using combinations of individual compound names and acronyms such as “benzalkonium chloride,” “BAC,” “BAK,” “didecyldimethyl ammonium chloride,” “DDAC,” “alkyldimethylbenzyl ammonium chloride,” “ADBAC,” “alkyltrimethylammonium chloride,” “ATMAC,” “cetylpyridinium chloride,” and “CPC” paired with “reproductive toxicity,” “reproductive effects,” “developmental toxicity,” “developmental effects,” etc. We identified exposure studies using search terms that paired “quaternary ammonium compounds” with “biomonitoring,” “exposure,” “dust,” “blood,” “urine,” “humans,” and “intake.” We also used citation mining to find studies cited by articles identified in our searches that our searches may have missed. Our goal was to include all studies in this area with limited exclusions. Studies with a financial conflict of interest, such as those funded by QAC manufacturers, are included in this review and noted as such. Studies not peer-reviewed or not published in English were excluded.

Human exposure to quaternary ammonium compounds

Toxicokinetics

Exposure to QACs can occur via inhalation, dermal contact, or ingestion, leading to chronic and acute effects over time. Evidence from rodent studies indicates that QACs are generally short half-life compounds that are excreted within hours of exposure [1, 14]. Metabolism and excretion are not yet well understood. Evidence from rodents suggests that excretion of QACs used as disinfectants occurs mostly in fecal matter, regardless of exposure route [1]. Benzalkonium chlorides (BACs) are oxidized on the alkyl side chain by cytochrome P450s into short-lived hydroxy compounds [15], followed by carboxylated metabolites, which have been measured in urine, whereas the BAC parent compound is measurable in feces [16]. Carboxylated BAC metabolites are the only QACs that are measurable in urine and could serve as a marker for regular biomonitoring despite lower concentrations compared to fecal excretion. In human samples, parent compounds, DDACs and ATMACs, were not measured in urine and appear in low levels in feces, suggesting that biotransformation is occurring, although sequestration in tissue is also possible [16]. A recent study generated and identified some metabolites of these classes in vitro using human liver microsomes, finding similar biotransformation to BACs, although some compounds may also undergo desaturation [17]. Overall, for most QACs, parent compounds are measurable in serum at low levels, but are quickly metabolized and excreted mostly in feces [1, 18]. However, more metabolites need to be quantified and standards made available for QAC biomonitoring to expand.

Exposure and biomonitoring studies

The EPA has expressed concern about the chemical residue remaining on surfaces after being cleaned and disinfected, which can lead to contamination of food and drinks humans consume [2]. An example of food contamination via processing was observed in a study of dairy samples [19]. Six types of QACs were detected in dairy milk, milking equipment, and storage containers disinfected with QACs. Assessment of the dairy milk samples collected during this study revealed that QACs, primarily C₁₂-BAC and C₁₆-BAC, were identified at concentrations ranging from 31.9 to 122 μg/kg [19]. Benzalkonium chloride and DDAC have been measured in infant formula samples and are attributed to contamination from processing, with concentrations in some samples exceeding 500 μg/kg [20]. In a study of vegetables obtained from an organic farm that used water and fertilizer free of QACs residues in Guangzhou, China, the QACs, cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium chloride (DTAC), and DDAC were measured in all of the vegetable samples but carrot with concentrations ranging from 23 to 180 μg/kg (dry weight) [21]. As these compounds have been detected in both sewage sludge and irrigation water and are predominantly used in personal care products, they are likely being taken up from the soil by plants.

Heightened concerns over increasing QAC exposure from disinfectants during the COVID-19 pandemic are supported by biomonitoring (Table 2), such as a study that found an increase in bioaccumulation of QACs in human blood following elevated use of products with QACs as the active ingredient during the pandemic [22]. In blood samples from Indiana before and during the COVID-19 pandemic, there was an overall 77% increase in QACs over 1 year, with median concentration increasing from 3.41 to 6.04 ng/mL. This study demonstrates that a rapid and dramatic increase in bioaccumulation of QACs in human blood occurred over a short period of time [22]. A similar study was conducted in 2021 in Virginia, in which blood samples were collected from a convenience sample of human subjects [23]. Of the 43 subjects, 35 presented with one or more types of QACs in their blood. Approximately half of the subjects had a total QAC concentration between 10 and 150 nM, which is comparable to concentrations found to be disruptive in in vitro models [24, 25]. Overall, these studies raise the concern that QACs can be detected in human blood with chronic exposure at levels that may be harmful to humans and other living organisms. Additional biomonitoring studies are needed since these studies had small sample sizes and were collected from a small geographic area, limiting the generalization of the findings to the general population. In addition, these studies were performed because the metabolism of QACs was well understood and may underestimate exposure due to quick biotransformation that reduces parent compound concentrations in serum levels.

Table 2.

Exposure studies

Type of QAC	Population/Sample	Study outcomes	Reference
ATMAC (C8–C22), BAC (C6–C18), DDAC (C8–C18)	46 dust samples derived from various locations (24 private dwellings, 16 public locations including offices, university auditoriums, and sport halls) in Flanders, Belgium	All 21 targeted QACs were detected in at least two dust samples. The median concentrations of individual QACs ranged between 0.00 and 2.99 μg/g. The three QACs with the highest median concentrations of 2.99, 1.86, and 1.64 μg/g were C12 BAC, C10 DDAC, and C14 BAC, respectively, which correlates to 26.8%, 15.6%, and 13.5% of the total QACs detected in the dust samples. Surprisingly, C22 ATMAC was an abundant QAC type found in the dust samples (not previously studied), with a median concentration of 1.45 μg/g, making up 10.8% of the total QAC concentrations calculated. Compared with US studies, the median concentrations of dust samples collected in Belgium were much lower, even pre-pandemic. The difference in these findings is likely due to different regulations for QACs between the USA and the EU.	[29]
ATMAC (C8–C18), BAC (C8–C18), DDAC (C8–C18)	111 dust samples from homes and public locations during the COVID-19 pandemic in South China	18 traditional QACs and 28 emerging QACs (9 BAC analogues, 10 DDAC analogues, 9 ATMAC analogues) were identified in dust samples. All traditional QACs were found in 71–100% of the dust samples. BACs were measured at the highest concentration, followed by ATMACs at and DDACs, which are different from the concentrations measured in the USA. Higher QAC levels were measured in rooms with carpets. Cinemas had the highest measured QAC concentration out of all the locations tested at 65.9 μg/g in cinemas, 58.3 μg/g in homes, 44.2 μg/g in offices, 34.2 μg/g in markets, 34.2 μg/g in hospitals, 28.4 μg/g in railway stations, and 23.7 μg/g in hotels.	[30]
BAC (C12–C16), DDAC	Human blood samples derived from 43 humans residing in a small college town; ⅔ college age and ⅓ older adults	Detection of one or more QAC was found in 35 out of 43 participants’ blood samples. 11 participants were found to have C12 BAC in their blood, 20 participants were found to have C14 BAC in their blood, 23 participants were found to have C16 BAC in their blood, and 32 were found to have C12 DDAC in their blood. Most participants had low concentrations of QAC with median concentrations of 1.9, 4.5, 2.5, and 5 nM for C12 BAC, C14 BAC, C16 BAC, and DDAC, respectively.	[23]
ATMAC (C8–C18), BAC (C6–C18), BAC metabolites, DDAC (C8–C18)	Human feces and urine samples collected from 14 donors, ages 8–60 from New York, USA	QACs in human feces were discovered at total concentrations ranging from 170 to 8270 ng/g dry weight. Concentrations of QACs in human feces were 2 orders of magnitude higher than in human serum (3.41–6.04 ng/mL), breast milk (1.5 ng/mL), and urine (0.49 ng/mL). This is suggestive that QACs are primarily excreted from the body via feces following exposure. BACs were the most abundant QAC type found in human feces and contributed to 49% of overall abundance, followed by DDACs making up 40% and BAC metabolites making up 9%. ATMACs were the least abundant, making up only 2%. The ratio of overall BAC metabolites to overall BAC in human feces (13.3%) was >10 times higher in comparison to the same ratio found in dog (0.3%) and cat feces (0.9%). These findings likely indicate higher biotransformation rates of BACs in humans than in cats or dogs.	[16]
ATMAC (C8–C18), BAC (C6–C18), BAC metabolites, DDAC (C8–C18)	10 human blood serum samples and 10 human urine samples, derived from New York City, USA	BACs, C14, C16, C18, and ATMAC C14 were detected in human blood serum at concentrations ranging from 0.28 to 3.40 ng/mL. Findings from this study confirm BACs, ATMACs, and DDACs are primarily found in human blood, while their metabolites are found primarily in urine. BAC metabolites, COOH-C6-BAC, COOH-C8-BAC, COOH-C10-BAC, COOH-C12-BAC, and OH-C12-BAC, were measured in the urine samples at average concentrations ranging from 0.05 to 0.35 ng/mL. COOH-C10-BAC was the most abundant metabolite, contributing to 35–46% of the total concentrations. For human serum, OH-C8-BAC and COOH-C10-BAC were detected at concentrations, 0.03–0.04 and 0.01 ng/mL, respectively.	[18]
ATMAC, BAC, BAC metabolites, DDAC	43 canine urine samples 36 canine feces samples 31 feline urine samples 26 feline feces samples from New York, USA	7 BACs, 6 DDACs, 6 ATMACs, and 8 BAC metabolites were measured in the samples. The median concentration of QACs in feces was found to be 9680 ng/g dry weight in dogs and 1260 ng/g dry weight in cats. BACs were the most abundant (64% of all QACs in dogs and 57% in cats). Samples collected from the animals in shelters contained higher levels of QACs than animals from homes; the exposure of pets in houses may be representative of human exposure.	[26]
ATMAC (C8–C18), BAC (C8–C18), ethyl BAC (mixture of C12 and C14), DDAC (C10–C16), benzethonium chloride (BZT), cetylpyridinium chloride (CTP)	Human liver microsomes (derived from 10 males and 10 females) 5 human fecal samples (2 samples from same participant and 3 samples from other participants), derived from adults aged 18–49 from Washington, USA	5/13 parent QAC compounds (C12 ATMAC, C12 BAC, C14 BAC, C16 BAC, and C10 DDAC) were detected in all 5 samples and 4 compounds (C18 ATMAC, C12 EtBAC, C14 EtBAC, and CTP) were identified in 80% (4 out of 5) of the samples. The relative abundance of BACs in 3 fecal samples collected that contained all 4 identified compounds was C10 < C16 < C12 < C14.	[17]
DDAC	Dust samples from approximately 2 m2 area in living room of 38 different families residing in Northern California, USA	DDAC was classified as a fungicide in this study and was detected in over 70% of the samples collected. The median concentration of DDAC in this study was found to be 2859 ng/g of dust.	[27]
ATMAC (C8–C18), BAC (C6–C18), DDAC (C8–C18)	Before COVID-19: 21 dust samples from residential homes in Indiana, USA During COVID-19: 40 dust samples from residential homes in Indiana, USA	All 19 QACs were detected in over 90% of the samples collected during the COVID-19 pandemic, and over 95% of the samples collected prior to the COVID-19 pandemic. The median QAC concentration of the samples collected during the pandemic increased by 62% when compared to samples collected pre-pandemic. BACs were the most frequently detected QAC type in the dust samples; specifically, C12 and C14 contributing to 29% of the overall QAC concentration. C10 and C18 DDAC were the most frequently found DDAC type in the dust samples, and C16 was the most frequently found ATMAC type in the dust samples, making up 10% of the overall QAC concentration. Households that cleaned more frequently had QAC concentrations 2× greater than those that did not.	[28]
ATMAC (C8–C18), BAC (C8–C18), DDAC (C8–C18)	Human liver microsomes and human blood serum samples (derived from 3 males and 3 females)	QAC concentrations in the blood collected during the COVID-19 pandemic were significantly higher compared to blood samples collected prior to the pandemic, and an overall increase in median concentration by 77%. Concentrations for BAC and ATMAC increased by 174% and 40%, respectively. For BACs, C12, C14, and C16, the samples collected during the pandemic were three times greater than in samples collected pre-pandemic.	[22]
ATMAC, BAC, DDAC	48 US lactating mothers	13/18 QACs were detected in breast milk collected. 7/18 QACs were present in over half of the samples. QAC concentrations present in breast milk ranged from 0.33 to 7.4 ng/mL (median 1.5 ng/mL). The most abundant QAC was BAC with a median concentration of 0.45 ng/mL. Suggests that QACs can transfer to children via breast milk and induce unwanted exposure to QACs	[33]

Recent studies have investigated the utility of measuring QACs in feces, as recent studies have identified it as the primary route of excretion. A method development study identified 35 QAC metabolites in five human fecal samples and quantified the concentration of C₁₀–C₁₆ BACs, identifying C₁₂ and C₁₄ as the most prevalent side chains with median concentrations of 217 and 320 nM [17]. These values are significantly higher than reported blood levels and suggest that blood measurements may be underestimated due to the toxicokinetic properties of QACs [24, 25]. Indeed, a recent study of paired human urine and fecal samples found median urine BAC concentrations of 0.49 ng/mL compared to 290 ng/g dry weight in feces, whereas ATMACs and DDACs were only measured in feces, not in urine. This study estimated the median cumulative daily intake for total QACs at 551 ng/kg [16]. A similar study from the same authors also measured QACs in urine and feces from cats and dogs and estimated median cumulative daily intakes for total QACs of 49.4 μg/kg BW/day for dogs and 4.75 μg/kg BW/day for cats, which are sentinel animals that share living environments with humans [26].

QACs have also been detected in household dust and these measurements were used to calculate estimated human intake. A 2019 study quantified the amount of various chemicals in consumer products found within household dust [27]. In this study, dust from 2 m² areas in the living rooms of 38 family homes in Northern California was collected. The primary QAC investigated in this study was DDAC, which is classified as a fungicide. The median concentration of DDAC in the dust was found to be 2.9 μg/g, about 15 times higher than the next most frequently detected fungicide. The high concentration of DDAC is attributed to its use as an antibacterial agent in household products. Worryingly, these samples were collected in 2015 and 2016 before the COVID-19 pandemic and likely significantly underestimate current dust levels. A more recent survey of dust in residential households in Indiana found an average of 62% increase in QAC concentrations in dust from before to during the pandemic, with median levels of total QACs rising from 36.6 μg/g (range: 6.55–127 μg/g) to 58.9 μg/g (range: 1.95–531 μg/g) [28]. A 2023 study of indoor dust from Belgium found a median total QAC concentration of 13.05 μg/g, which is lower than samples from the USA but the pattern of individual compounds was similar to US measurements [29]. Conversely, a pandemic-era study in China found media dust concentrations of 58.3 μg/g in homes, 65.9 μg/g in cinemas, 44.2 μg/g in offices, 34.2 μg/g in markets, 34.2 μg/g in hospitals, 28.4 μg/g in railway stations, and 23.7 μg/g in hotels, with carpeted rooms and environments having higher concentrations than non-carpet rooms [30].

Exposure in sensitive populations

There are significant disparities in QAC exposure between men and women. Women are more likely to be exposed than men due to frequent use of cleaning, personal care, and cosmetic products [31, 32]. In addition, there are concerns about differences in reaction to exposure via inhalation in women compared to men. A 2018 study found that women exposed to a mixture of cleaning supplies due to combined work and household use were more likely to experience a decline in lung function compared to men who experienced similar exposures [32]. It is possible that women are more at risk of chronic exposure to QACs and therefore, more likely to experience QAC-related health effects. Additional research into sex differences in both exposure and response is necessary to better understand their health impacts.

The Indiana dust study calculated estimated daily intakes (EDIs) of BAC and DDAC in both adults and children throughout the COVID-19 pandemic, finding the highest EDI of QACs in toddlers residing in homes that disinfect frequently to be 10 times higher than the EDI of QACs by adults [28], but EDIs for these QACs are below the tolerable daily intake threshold of 1 × 10⁵ ng/kg/day declared by the European Food Safety Authority (EFSA). This study stresses that children may be more susceptible to the effects of QACs than adults in households that disinfect with QACs often, given the significant disparities in their EDIs.

Another rising concern for QACs is exposure during critical periods of development. Between March and October 2019, breast milk samples collected from 48 US mothers contained 13 of 18 targeted QACs, with 7 of the QACs present in over half of the samples [33]. The concentrations of QACs in the milk ranged from 0.33 to 7.4 ng/mL (median 1.5 ng/mL). The most abundant QAC was BAC, with a median concentration of 0.45 ng/mL. Overall, this study suggests that QACs can transfer to infants via breast milk.

With limited knowledge of human exposure levels and the concentration of these chemicals within our bodies, it is difficult to assess the relevancy of existing studies in mammalian models to human exposure. Additional biomonitoring is warranted but must consider knowledge of biotransformation. The National Health and Nutrition Examination Survey (NHANES), a program from the US Center for Disease Control that was started in the 1960s in order to survey the overall health of American citizens by measuring the concentrations of hazardous chemicals, does not yet measure QACs [34]. Including oxidized BAC metabolites in NHANES would improve the generalizability of exposure studies in the US population [16]. However, for other QACs, more characterization of toxicokinetics is necessary before biomonitoring can be expanded.

Reproductive & developmental effects of quaternary ammonium compounds in animal studies

Female reproductive toxicity

Since women have elevated exposure to QACs due to their daily use of cleaning and cosmetic products, it is essential to explore the impacts of QACs on female reproductive function. Exposure to everyday compounds is proposed to be a contributing factor to declining fertility rates in recent years [35]. While the harmful impacts of QACs are a rising public health concern, few studies have investigated their possible impacts on female reproduction, with existing studies largely focused on acute exposure in rodent models [36]. Female, male, and developmental toxicity studies are summarized in Table 3.

Table 3.

In vivo studies

Type of QAC	Dose	Model	Study outcomes	Reference
BAC	120 mg/kg body weight/day d7-BAC C-16 for 1 week	Adult male and female C57BL/6J mice	Decrease in lanosterol and sterol metabolites is indicative that BACs inhibit cholesterol biosynthetic pathway as well as lipid and sterol homeostasis in neonatal brains. With extended exposure and accumulation of BACs in the body, it can have significant effects on DNA transcription and sterol/lipid profiles. This confirms that BAC is a possible developmental neurotoxicant.	[44]
ADBAC, DDAC	Rats:   0, 10, 30, or 100 mg/kg/day for ADBAC 0, 1, 10, or 20 mg/kg/day DDAC Rabbits: 0, 1, 3, or 9 mg/kg/day for ADBAC 0, 1, 3, or 10 mg/kg/day DDAC	Pregnant CD rats New Zealand white rabbits	Mean maternal body weight decreased in rats at 20 mg/kg/day during dosing period and in rabbits at both 3 and 10 mg/kg/day during dosing period. All external, visceral, and skeletal prenatal developmental malformations were determined to be uncorrelated with QAC treatment. *Study was sponsored by the ADBAC & DDAC Issues Steering Committees, under the guidance of Household and Commercial Products Association	[51]
ADBAC, DDAC	ADBAC:   0, 300, 1000, or 2000 ppm DDAC: 0, 300, 750, or 1500 ppm	Male and female rats of 2 generations (parents and offspring)	Body weight of offspring pups were significantly reduced after being dosed with 2000 ppm of ADBAC on postnatal days 14–28. Body weight of adult rats were noted to decrease at doses 2000 ppm ADBAC and 1500 ppm DDAC. *Study was sponsored by the ADBAC & DDAC Issues Steering Committees, under the guidance of Household and Commercial Products Association	[52]
ADBAC + DDAC	8 weeks fed at 60 or 120 mg/kg/day oral gavage at 7.5, 15, or 30 mg/kg	CD-1 mice and Sprague–Dawley rats	At day 18 of gestation, developmental defects were witnessed in offspring including split face lesions, cranial lesions, spinal lesions, and abnormal phenotype. With the highest dose, 120 mg/kg/day, fetal death and placental abnormalities were observed. Study suggests that even with either maternal or paternal exposure to QACs, it is enough to cause neural tube defects in offspring. These neural tube defects can persist across two generations. Neural tube defects were witnessed in all dosed groups of mice/rats and most prevalent when both parents were exposed to the QAC mixture.	[46]
Tetrabutyl ammonium bromide (TBAB)	0.125–35 mg/L Over 24 h	Zebrafish embryos	Short-term exposure to TBAB resulted in impaired brain development (cranial neural crest), loss of midbrain-hindbrain, abnormal tailbud development, anterior–posterior axis shortening, and inner ear defects. Other effects observed from TBAB exposure included eye deformities, axis abnormalities, and pericardial edema. Hatch rate of embryos decreased with increasing TBAB concentrations. The mortality rate of the zebrafish embryos was directly proportional to the TBAB concentration increasing.	[45]
CPC	0.12 and 1.2 mg/kg bw/day; pregnant mice exposed for 3 days	Fertile male and female CD-1 mice	The total number of oocytes decreased in treatment groups and was observed as overall oocyte loss in the neonatal ovaries. At a dose of 1.2 mg/kg, reactive oxygen species–positive oocytes increased, causing increased expression of gamma-H2AX protein, which is linked to cell oxidative stress and further causes DNA damage. A 1.2 mg/kg reduced morula formation/development was observed, causing impaired development of embryos during the fetal stage. Exposure to CPC reduces mitochondrial function in oocytes and the development of viable blastula. Exposure to CPC potentially compromises female fertility during pregnancy.	[49]
Ethyl(dimethyl) (tetradecyl) ammonium ethyl sulfate	0, 5, 15, or 50 mg/kg	7-week old Sprague–Dawley male and female rats	At a dose of 15 mg/kg/day, relative ovary weight in female rats decreased significantly by end of dosing, but was not dose-dependent. There were no changes to estrous cycle, population, length of gestational period, or length of conceiving in female rats.	[41]
ADBAC + DDAC	0, 60, 120, 240, and 480 mg/kg	Female CD-1 mice	Mice receiving doses, 240 and 480 mg/kg, lost an appetite, became lethargic, and developed a rough haircoat. Euthanasia of pregnant dams was needed for those treated with 60 and 120 mg; symptoms include lack of appetite, lethargy, ataxia, kyphosis, labored breathing, cyanosis, vaginal hemorrhage, and dystocia. Mice that received 0 mg of the disinfectant mixture appeared normal and healthy throughout the duration of the study. Time to first litter was longer in breeding pairs dosed with 120 mg vs. 0 and 60 mg. At this dose, there was also a reduced number of pups per litter and/or dam mortality.	[38]
ADBAC + DDAC	120 mg ADBAC + DDAC/kg/day for 2 or 8 weeks for male mice exposed through routine disinfectant use for 7 weeks and/or 7.5 mg ADBAC + DDAC/kg body weight for 8 days	Male and female CD-1 mice	Data suggests that ADBAC + DDAC exposure at these doses are toxic to both male and female fertility. Female mice exposed to ADBAC + DDAC experienced lower reproductive capacity with lack of ovulation and fewer cycles of estrogen. Male mice exposed to ADBAC + DDAC experienced a significant decrease in sperm concentration and motility. More research needed to be conducted to determine the mechanism(s) on how these QACs disrupt female and male reproduction.	[39]
CPC	(0, 0.5, 1.0, 1.5, 2.0, and 3.0 μM; 0, 0.18, 0.36, 0.54, 0.72, and 1.07 mg/L)	B. orientalis (amphibian model)	The survival rate of embryos decreased in a concentration-dependent manner. After 168 h (7 days of treatment) of exposure, the survival rate dropped at 2 μM and LC50 was noted to be 1.95 μM. At a dose of 1.5 μM and time point, 168 h, malformations increased in embryos including abnormal gut coiling, head malformation/dysplasia, and hematoma. At a dose of 1.5 μM, body + tail lengths of embryos were shorter than the control group. At a dose >2 μM, shortened tail muscle height and reduced eye diameter in embryos were observed At a dose of 2 μM, the survival rate of tadpoles significantly decreased.	[48]
BAC	0, 0.1, 0.5, and 2.5 mg/L	Zebrafish embryos	At doses, 0.1, 0.5, and 2.5 mg/L, hatching delays, mortality, tail malformations, and pericardial edema were observed over the course of 72 h. At a dose of 2.5 mg/L, mortality increased during the first 48 h of exposure and displayed more “anxious” and “erratic swimming” behaviors.	[47]
BAC	0.1–0.5 mg/L	Adult, wild-type zebrafish and C. elegans embryos	At dosing range, 0.1–0.5 mg/L, there was concentration-dependent mortality of zebrafish. At a dose of 0.5 mg/L, the mortality rate of zebrafish increased significantly by ~40% (48–81%). Delayed hatching was observed in the first 48 h of exposure. BAC was noted to be the most toxic among three replacement compounds, with greater toxic effects on embryo hatch rate and mortality; toxicity is comparable to triclosan.	[43]
Benzyldimethyldodecyl ammonium chloride Methylbenzethonium chloride	100 μM	C. elegans	At 100 μM, greater than 20% embryonic lethality was observed, which is suggestive that these QACs cause reproductive toxicity. No significant difference in overall bioactivity, but bioactivity is high for methylbenzethonium chloride.	[61]
Tetradecyltrimethyl ammonium bromide (TTAB) Tetradecyltrimethyl ammonium chloride (TTAC)	0.88 mg/L	Wild-type N2 C. elegans	Results revealed oscillatory reproductive effects over the course of four offspring generations; stimulation of C. elegans was observed in F1 and F3, while inhibition was observed in F2 and F4 with exposure to TTAC. With exposure to TTAB, stimulation was observed across all four generations with the greatest stimulation in F2. The varying findings between TTAC and TTAB suggests that the halogen anion influences QAC toxicity. Both biochemical and gene expression data support that exposure to TTAC and TTAB impacted oocyte meiosis, gonadal support, and germline development.	[42]

The potential female reproductive toxicity of environmental exposure to QACs was discovered by accident. In 2005, Dr. Patricia Hunt at Washington State University discovered a fertility issue in her newly relocated rodent colony. Only 10% of her female mice achieved pregnancy, and even fewer produced live offspring, a significantly lower rate than before moving the colony. A little scientific detective work traced these effects to the disinfectant product that was used to clean the mouse cages. This product included a mixture of two commonly used QACs: ADBAC and DDAC [13]. This was not the first time Dr. Hunt connected reproductive defects to a synthetic chemical. In 2003, she similarly identified egg defects in her mice from bisphenol A (BPA) exposure [37]. Bisphenol A can leach from plastics and has since been outlawed in baby products. Like BPA, QACs are widely used daily, resulting in chronic, low-dose exposure.

The discovery of QACs inhibiting fertility in the animal facilities at Washington State University, and later at Virginia Tech, led to two academic studies on the developmental and reproductive toxicity of an aqueous mixture of the two implicated disinfectants, 6.67% ADBAC and 10.1% DDAC [38, 39]. In both studies, mice were bred for two generations in a QAC-free facility to minimize background contamination. To assess chronic effects, mice were exposed to the mixture at concentrations of 0, 60, or 120 mg/kg/day through diet starting at 6 weeks of age for a total of 6 months. Overall, fertility was decreased in the exposed pairs. Female mice dosed at 120 mg/kg/day experienced significantly longer time to first litter, fewer pregnancies in the first 100 days of exposure and fewer pups per litter than those exposed to 0 or 60 mg/kg/day.

A follow-up study assessed the sex-specific effects on adults dosed with 120 mg/kg/day [39]. Females exposed to the mixture for 8 weeks before and during breeding with an exposed male experienced significantly fewer estrous cycles and less ovulation during the study period, leading to lower reproductive capacity. No significant differences existed between the number of corpora lutea found in the control group and a group dosed for 2 weeks. However, the combination of mice inhabiting the QAC-exposed environment and dosed with QACs for 8 weeks had fewer viable embryos, suggesting that long-term, ambient exposure QACs could pose a risk to human fertility. The authors state that additional research is needed to identify the mechanism through which the mixture acts, hypothesizing that an endocrine-disrupting mechanism could be at play, meaning they interfere with normal hormonal function [40].

In a regulatory study in which young adult Sprague–Dawley rats were treated via gavage with 0, 5, 15, or 50 mg/kg of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate, used in soaps and personal care products, from 2 weeks before mating through lactation, treated females exhibited decreased relative ovary weight in the 15 mg/kg treatment group. This finding was dismissed due to non-monotonicity, despite the potential for an endocrine-mediated mechanism of action, and was not followed up [41].

Given that the animal facility findings and follow-up studies are nearly 10 years old, it is surprising that no other groups have investigated the female reproductive toxicity of QACs. Additional research, including at low doses and using a variety of individual compounds and mixtures, will be necessary to understand the impact of exposure to QACs in daily life.

Male reproductive toxicity

Few studies have investigated the effects of QACs on male reproductive function in animal models. A study following up the animal facility contamination at Virginia Tech assessed reproductive effects in males following either ambient exposure to daily disinfection practices in the animal facility or 8 days of oral gavage of the ADBAC + DDAC mixture [39]. Both the mice exposed ambiently and those dosed through oral gavage experienced a significant decrease in sperm concentration and motility. The viability of sperm from exposed male studs in breeding experiments, in which males were exposed to 120 mg/kg/day via diet for 8 weeks, was not significantly impacted, although fecundity was decreased. This finding suggests that at high-dose exposure, sperm viability is not a mechanism of ADBAC + DDAC-induced subfertility. However, the lower dose and ambient exposure experiments had more significant effects on sperm quality, suggesting that further research is needed using low doses representing human exposure in daily life. Quaternary ammonium compounds have also been shown to impact proteins necessary for sperm function and fertilization in Caenorhabditis  elegans [42].

Developmental toxicity

Multiple studies have identified a link between prenatal exposure to QACs and neurotoxicity. In zebrafish larvae, BAC and BEC exposure induced alterations in secondary motor neuron axonal projections, an indicator of neurotoxicity [43]. Benzalkonium chloride has been shown to cross the blood–placenta barrier and localize into the embryonic brain in mice, resulting in gene expression changes and disruption of lipid homeostasis in the brain [44]. Tetrabutylammonium bromide has also been shown to disrupt brain development in zebrafish embryos [45]. Neural tube defects in rodents have been observed following ambient vivarium exposure and in offspring of rodents orally dosed during pre-conception and gestation [46]. In addition, fetuses from both groups had statistically significantly lower fetal and placenta weight compared to controls. A species sensitivity difference was observed, with mice exhibiting a higher incidence of neural tube defects than rats under the same conditions. Neural tube defects were not found to persist transgenerationally after exposure was removed. However, experiments in which only one parent was exposed revealed no sex differences; exposure of either parent is sufficient to induce neural tube defects. Importantly, ambient exposure during gestation resulted in significantly increased levels of neural tube defects compared with unexposed controls, indicating that ambient exposure to residue from cleaning constitutes a significant risk [39]. These findings suggest that QACs are teratogens and may pose a significant concern during pregnancy, especially for individuals who regularly use disinfectants. However, the neurotoxic potential of QACs typically used in personal care products is still unknown.

The authors of the ambient exposure studies noted that keeping control animals in another room at the same facility as animals being dosed with QACs was insufficient to maintain unexposed controls, even if QAC disinfectants were not used, because facility-wide animal husbandry, such as central cage washing, resulted in contamination of control cages with QACs. This highlights the challenges of creating unexposed control groups for chemicals with ubiquitous use in daily life, as well as the risk to humans from residue from cleaning products.

BACs and other disinfectants have been identified as developmental toxicants in rodents and non-mammalian animal models, including zebrafish and worms. A 2018 study compared the toxicity of legacy disinfectants, triclosan and triclocarban, to replacements, such as BAC and benzethonium chloride (BEC) [43]. In this study, replacement chemicals were not found to be any less developmentally toxic than triclosan and triclocarban to zebrafish and C. elegans [43]. Benzalkonium chloride exhibited the greatest toxicity toward zebrafish embryos, with acute lethality occurring in the range of concentrations detected in municipal wastewater (ng/mL). In another zebrafish study, embryos were exposed to 0.1, 0.5, and 2.5 μg/mL of BAC for up to 96 hours [47]. After 48 h of exposure, embryo mortality, delayed hatching, and tail malformations were observed at the highest concentration of BAC. Additionally, anxiety and erratic swimming were observed after 96 h of exposure.

Beyond BACs and disinfectants, a few studies have assessed the developmental toxicity of cetylpyridinium chloride (CPC), a commonly found QAC in lotions, deodorants, and oral antiseptic and hygiene products, such as mouthwash, toothpaste, and breath/throat sprays. Using the amphibian model Bombina orientalis [48], embryos were exposed to 0.18, 0.36, 0.54, 0.72, or 1.07 μg/mL of CPC. Developmental defects observed in the tadpoles included abnormal gut coiling, head malformation, and hematoma. The body and tail length of the tadpole embryos were significantly shortened after treatment with 0.54 μg/mL CPC, as well as shortened tail muscle height and reduction of eye diameter after treatment with 0.72 μg/mL CPC. Cetylpyridinium chloride has also been assessed in a rodent model. Ovaries from 1 and 3 day-old pups that were developmentally exposed to 0.12 or 1.2 mg/kg CPC from days 16.5 to 18.5 of pregnancy exhibited reduced size and oocyte loss [49]. Mitochondrial damage and dysfunction observed in oocytes persisted into adulthood and were observed alongside decreased levels of estradiol, decreased anti-Mullerian hormone production, and increased rates of oocytes with DNA damage markers. Embryo development was significantly impaired after prenatally exposed females were mated with unexposed males. This finding is important because disruption of ovarian development during the prenatal window is irreversible. The pool of oocytes cannot be renewed, and its premature depletion can lead to decreased fertility, premature ovarian failure, and hormone-mediated diseases later in life. Importantly, this study was conducted at doses representative of human exposure levels.

In contrast, industry and government studies of various QACs have largely identified minimal developmental toxicity, although many of these studies from the late 20th century are not publicly available [50]. We identified a handful of recent peer-reviewed studies performed in rats and other mammalian models. When young adult Sprague–Dawley rats were treated via gavage with 0, 5, 15, or 50 mg/kg of ethyl(dimethyl)(tetradecyl)ammonium ethyl sulfate, used in soaps and personal care products, from 2 weeks before mating to lactation, no effects on gestation or development were observed [41]. Reduced food intake was noted after day 2 of exposure for the 50 mg/kg treatment group in both sexes. In a study sponsored and funded by the Household and Commercial Products Association, CD rats and New Zealand white rabbits were exposed to ADBAC and DDAC via gavage from gestation day (GD) 6–15 for rats and GD 6–18 for rabbits [51]. Reduced food intake and weight loss were observed in rabbits dosed with 10 mg/kg of ADBAC and ≥3 mg/kg DDAC and rats dosed with ≥12.5 mg/kg DDAC. No teratogenic effects of ADBAC or DDAC were observed in this study. Overall, rabbits were more sensitive than rats to exposure. In another study from the same group, CD rats were exposed via diet to approximately 15–120 mg/kg ADBAC or DDAC for two generations [52]. No treatment effects on fertility were observed, although prenatally exposed pups in the highest treatment groups exhibited reduced body weight. Overall, these studies find that the tested QACs require much higher doses than humans are exposed to for reproductive toxicity to occur. However, they focused on survival, body weight, and gross birth defects and did not evaluate any tissue, cellular, or molecular effects.

Reproductive, developmental, & endocrine effects of quaternary ammonium compounds in in vitro studies

In vitro studies of QACs provide support for an endocrine-mediated mechanism of action, although the exact mechanism is still unclear (Table 4). In a study evaluating estrogenic activity, recombinant human breast carcinoma cells were used to assess the potential of CPC and BAC to act as endocrine disruptors [24]. Both CPC and BAC exhibited antiestrogenic activity with half-maximal effective concentration (EC₅₀) of 4.5 μM (1.5 μg/mL) for CPC and 17.5 μM (5.5 μg/mL) for BAC. No estrogenic activity was observed. An estrogen and androgen receptor transactivation study of DDAC and ADBAC did not identify activation at doses below cytotoxic levels (0.1–2.5 μg/mL), although this study only assessed the chemicals individually. In another in vitro analysis, exposure to 1.0–1.5 μg/mL of BAC significantly increased estrogen biosynthesis in the H295R steroidogenesis assay [53]. In addition, exposure to BAC significantly increased gene expression of enzymes involved in steroidogenesis, including transcripts for HSD3B1, HSD17B1, HSD17B4, and CYP19A1. Benzalkonium chloride has also been shown to inhibit the biosynthesis of cholesterol, a precursor of sex steroid hormones [25]. In estrogen-dependent breast cancer MCF-7 cells, exposure to 0.5–1.5 μg/mL BAC significantly increased cell proliferation, suggesting an estrogenic mechanism of action. Benzalkonium chloride exposure also increased levels of estrogen receptor alpha (ERα) and G-protein-coupled estrogen receptor 1 (GPER1), as well as altered cell cycle progression.

Table 4.

In vitro studies

Type of QAC	Dose	Model	Study outcomes	Reference
BAC, CPC	0.13–26.7 μM	Human breast carcinoma MCF-7-derived VM7Luc4E2 and ERalpha-positive cells	Dose–response curves of two assays revealed that CPC’s antiestrogenic activity results in CPC-mediated inhibition of the mitochondrial electron transport chain and reduces mitochondrial activity or encourages mitotoxic effects. Both BAC and CPC inhibit mitochondrial complex 1 and display antiestrogenic activity in vitro at low (micromolar) concentrations that may be physiologically relevant.	[24]
BAC	24–48 h 10 μM, 5 μM, 1 μM, 100 nM, and 10 nM	Mouse neuro2a and human SK-N-SH neuroblastoma cell lines	BACs can potently inhibit the cholesterol biosynthesis enzymeDHCR7, and exposure to BACs can have severe consequences despite no apparent acute toxicity. The inhibition of cholesterol biosynthesis by BACs could potentially be responsible for deleterious effects on embryonic development.	[25]
BAC (mixture of C12, C14, and C16)	1.0–1.5 mg/L	Human adrenal H295R cell line	At a dosing range of 1.0–1.5 mg/L, E2 levels were significantly increased in cells, and biosynthesis increased by 1.73-fold and 2.22-fold, respectively. At a dose of 1.5 mg/L, transcription of genes, 3beta-HSD2, 17beta-HSD1, 17beta-HSD4, and CYP19A, were significantly increased by 1.8-fold, 2.3-fold, 2.1-fold, and 2.0-fold, respectively.	[53]
C12 and C14 alkyl(ethylbenzyl) dimethylammonium, BAC, barquat	0, 0.5, and 5.0 μg/mL 5 days of exposure	Rat pheochromocytoma cell line PC-12 cells	All three QAC types did not induce neurite outgrowth.	[62]

Effects of quaternary ammonium compounds in humans

Epidemiological studies of the associations between QAC exposure and health endpoints are sorely lacking (Table 5). One study assessed the impact of ambient exposure to QACs in humans. A study conducted by Hrubec et al. in 2021 collected human blood samples to study any endocrine-disrupting effects of ADBAC, BAC, and DDAC on these samples [23]. Concentrations of the three chemicals of interest were quantified, and the blood samples were tested for biomarkers of mitochondrial function, sterol pathways, and cytokine analysis. Overall, cholesterol synthesis intermediaries were significantly disrupted, similar to previous studies, with different markers altered by DDAC compared to BAC. In addition, mitochondrial function was decreased, and inflammatory cytokines were increased in samples with high QAC levels.

Table 5.

Human toxicity studies

Type of QAC	Dose	Model	Study outcomes	Reference
ADBAC, BAC, DDAC	Between 10 and 150 nM	Human blood samples derived from 43 humans residing in a small college town; ⅔ college age and ⅓ older adults	From human blood samples collected to test biomarkers such as mitochondrial function, sterol pathways, and cytokine analysis, results revealed that half of the models having total QAC concentrations between 10 and 150 nM had physiological effects in cell culture models. A rise in the lanosterol level is indicative of a decrease in a downstream step of the cholesterol synthesis pathway or a general upregulation of cholesterol production.	[23]
BAC, TDBAF	Serial dilutions (1/50, 1/60–1/260) of TDBAF, derived from 0.1% stock solution of the compound (2.84 mM) 10 s, 2, 4, 6, 10, 20, and 30 min	Adult men, ranging from 22 to 45 years old	EM revealed drastic changes in density and appearance of sperm chromatin after sublethal treatment with TDBAF such as the chromatin appearing more granular and less condensed than in control spermatozoa. Sperm of DNA is susceptible to change with exposure to TDBAF and a possible risk factor for reproductivity.	[55]

The spermicidal properties of QACs have been known since the 1970s, and BACs are widely used in commercial spermicide formulations worldwide [54]. Quaternary ammonium compounds act as detergents, disrupting the lipid bilayer and eventually disabling sperm motility. Despite the widespread use of QACs, specifically for their reproductive toxicity in this context, little attention has been paid to the potential for similar effects or endocrine-disrupting mechanisms at low concentrations from ambient exposure. Quaternary ammonium compounds are used in spermicides at concentrations known to be lethal to sperm, typically 0.01% (100 ng/mL). Concern over the possibility of exposure to sub-lethal concentrations damaging sperm without preventing contraception, which could cause adverse fetal outcomes, led to a study of the impact of low doses of QACs on sperm [55]. The benzalkonium homolog, tetradecyl-dimethyl-benzyl-ammonium fluoride (TDBAF), was used as a model QAC. Semen samples from 32 fertile men, ages 22–45, were exposed to TDBAF at concentrations where sperm were still motile (as low as 0.0001% or 1 ng/mL). Overall, the study found that QACs did not damage DNA or induce the production of reactive oxygen species (ROS) at sublethal concentrations. However, follow-up studies using semen samples from infertility patients found that exposure to 0.001% (10 ng/mL) TDBAF caused a statistically significant decrease in ROS levels. In addition, exposure to 0.0001% (1 ng/mL) TDBAF, comparable to QAC levels in human blood from ambiently exposed Americans, caused a more modest in magnitude, but statistically significant increase in ROS levels [55]. Thus, TDBAF exhibits a non-monotonic dose–response at low doses. Reactive oxygen species–damaged sperm are associated with male infertility; these results suggest that subfertile or infertile males may be susceptible to exacerbation of problems with sperm health from ambient exposure to QACs.

Future directions

Although few studies have been performed analyzing the reproductive and developmental toxicity of QACs, the existing evidence that low-dose exposure to QACs may decrease fertility, damage sperm, and disrupt development, possibly through an endocrine-mediated mechanism, emphasizes the need for further study (Figure 1). It is especially important to thoroughly investigate the possible reproductive and developmental toxicity of QACs because of the sensitivity of endocrine signaling and developmental processes during critical windows.

Figure 1.

Summary of the adult male and female (A) and developmental (B) effects from QACs described in this review, mostly from animal studies.

Low doses that represent human exposure are one area in which more studies are needed. Biomonitoring and EDI estimates suggest that human intake is orders of magnitude lower than many studies described in this review. Due to the potential of QACs to act as endocrine-disrupting compounds and exhibit non-monotonicity, high-dose studies cannot be construed to represent estimated human exposure levels. Biomonitoring and exposure assessment also need to be expanded to provide better insight into true human exposure levels, which will require greater knowledge of QAC toxicokinetics, especially biotransformation and excretion pathways. Once the likelihood of metabolites to reach organs and tissues is confirmed, in vitro studies of metabolites will be necessary to determine to what extent biotransformation results in activation to toxic metabolites.

The reprotoxicity and embryotoxicity studies included in this review are mostly in vivo rodent studies. Although mice are useful for modeling reproductive processes, it is unknown if the mechanisms by which QACs disrupt mouse reproductive systems are parallel to humans. Additional studies related to reproductive and embryotoxicity in additional models are needed to understand the safety, adverse effects, and mechanisms by which QACs act on these target human systems. Sex-specific effects from developmental exposures have not yet been assessed.

In addition, the evidence discussed herein suggests that the toxicity of QACs may vary significantly based on species, route of exposure, dose, and endpoints assessed. When assessed side-by-side in the same study, rats were less sensitive to the same doses of QACs compared to mice [46]. The insensitivity of rats to various doses that disrupt reproduction in mice or humans is well documented for the female reproductive system, yet many regulatory studies exploring potential endocrine disrupting chemicals continue to utilize rats [56, 57]. Importantly, “no effect” findings in rat studies do not negate the importance of findings in other models [58]. Future studies need to consider species sensitivity differences.

Most studies described in this review relied on oral dosing, but oral exposure is not the most plausible route of human exposure to QACs. Both dermal contact and inhalation exposure result from using QAC-containing disinfectants or being exposed to objects or places where QAC disinfectants were previously used. Importantly, in studies where rodents were ambiently exposed via the animal facility, the doses were not quantified, but the effects from ambient exposure compared to rodents raised in a non-QAC facility were more significant than animals exposed only via oral dosing. This may be due to quick first-pass metabolism from oral dosing that is bypassed in other routes of exposure [23]. Studies that model inhalation exposure, which has a higher bioavailability than oral exposure, are necessary to better model human exposure.

Distinct differences in the findings of academic and regulatory studies are evident. Although many regulatory studies are unpublished, the few recent available studies focused mainly on very high doses and gross morphological endpoints in rats, whereas academic studies utilized a broader range of species, doses, and endpoints, including molecular analyses of hormone levels and gene expression changes. This is typical in the field of endocrine disruption research and was one of the driving factors for the design of the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA) project [59]. Existing studies suggest an endocrine-mediated mechanism of action for QAC effects in reproduction, although additional testing is necessary.

QACs are a complex class of compounds. Most named compounds have multiple side chain lengths, often used in products as a mixture. As a result, there are hundreds of QACs in commerce, yet the studies described herein focus on a few major compounds used in common disinfectants. Additionally, some studies do not identify or report the side chains investigated. One type of QAC that we did not identify any studies on, despite its popularity in consumer products, is behentrimonium methosulfate (BTMS). Behentrimonium methosulfate is found in cosmetic and personal care products, such as lotions and hair care products. None of the studies presented in this review study BTMS as a potential reproductive toxicant. This is just one QAC that we have identified that requires more research on its potentially toxic effects.

Mixture effects between QACs and other compounds, such as pharmaceuticals, are also widely understudied. In a 2018 study, binary mixtures of BAC and select anticancer drugs such as 5-fluorouracil, cisplatin, etoposide, and imatinib mesylate were evaluated for reprotoxic and genotoxic effects using the freshwater crustacean, Ceriodaphnia dubia [60]. Interestingly, treatment with mixtures produced antagonistic effects (less toxicity) at some doses compared to either compound independently. This study used environmentally relevant low dose treatments; further studies should identify if the presence of ubiquitous QACs can interfere with prescribed drugs or interact with other environmental exposures.

Overall, little is known about the potential reproductive toxicity of most QACs. Future studies should employ sensitive animal models and environmentally relevant doses and routes of exposure to assess a broad range of QACs and mixtures to help understand the all-inclusive health impacts of QACs. Conducting similar studies with their possible alternatives is essential to break the cycle of regrettable substitutions. Proactive product ingredient testing for alternatives should be developed to ensure that products available are safe for human and environmental health.

Conclusion

From the studies included in this review, it is evident that further testing is needed to ensure that QACs are not detrimental to reproductive and developmental health in both sexes. Human use of QACs has expanded significantly following the removal of triclosan from consumer products and the COVID-19 pandemic, and the limited available biomonitoring studies reflect this increase in exposure. Studies in rodents and non-mammalian models suggest that QACs may be endocrine-disrupting chemicals. Given the limited information on QACs, it may be prudent to take a precautionary approach to their usage in schools and daycares, where regular disinfection procedures are performed, and children are likely to be exposed. Further studies on QAC toxicity as well as biomonitoring, toxicokinetics, and ecological risk are necessary to fully understand the potential risk of increasing QAC exposure.

 

Conflict of interest: The authors have declared that no conflict of interest exists.

Author contributions

LB, AH, and GRW conceived the idea, researched the topic, and wrote and edited the review.

Data availability

Data sharing not applicable – no new data were generated.