Potential Developmental and Reproductive Impacts of Triclocarban: A Scoping Review

Triclocarban (TCC) is an antimicrobial agent used in personal care products. Although frequently studied with another antimicrobial, triclosan, it is not as well researched, and there are very few reviews of the biological activity of TCC. TCC has been shown to be a possible endocrine disruptor, acting by enhancing the activity of endogenous hormones. TCC has been banned in the US for certain applications; however, many human populations, in and outside the US, exhibit exposure to TCC. Because of the concern of the health effects of TCC, we conducted a scoping review in order to map the current body of literature on the endocrine, reproductive, and developmental effects of TCC. The aim of this scoping review was to identify possible endpoints for future systematic review and to make recommendations for future research. A search of the literature until August 2017 yielded 32 relevant studies in humans, rodents, fish, invertebrates, and in vitro. Based on the robustness of the literature in all three evidence streams (human, animal, and in vitro), we identified three endpoints for possible systematic review: estrogenic activity, androgenic activity, and offspring growth. In this review, we describe the body of evidence and make recommendations for future research.


Introduction
Triclocarban (TCC) is an antimicrobial agent primarily used in bar soaps, plastics (including toys, tableware, and utensils), cleansing lotions, and wipes [1,2]. Although TCC and triclosan are frequently discussed and studied in tandem, TCC is a carbanilide (the two aromatic rings are linked by urea), whereas triclosan is a diphenyl ether (with oxygen linking the rings) [3]. Because of this, they may have different biological activity [3,4].
Due to concerns regarding the effects of TCC on human health, exposure has been assessed in human populations residing in China, Europe, Canada, and the US. In the large-scale US biomonitoring study NHANES (2013-2014), TCC was detected in human urine in ∼36% of individuals with concentrations up to 68 g/L. Sex (male), age (20+ years), race (non-Hispanic black), and increased body surface area were significantly associated with higher concentrations of TCC [5]. In another US study of pregnant women in Brooklyn, TCC had a detection frequency of 86.7% and its metabolites 2 -OH-TCC, 3 -OH-TCC, and 3 -Cl-TCC were found in 27.1%, 16.6%, and 12.7%, respectively [6]. In a study done by the US Centers for Disease Control (CDC), TCC was detected in 35% of urine samples tested, and the metabolites 2 -OH-TCC and 3 -OH-TCC were detected in 16% and 14.5% of urine samples, respectively. In the same study, serum TCC was detected in 44% of samples (although the metabolites were not detected above the limit of detection (LOD)). TCC was again detected more frequently in males than in females in the CDC study [7].
Interestingly, a Canadian biomonitoring study (the Canadian Health Measures Survey [CHMS]) found that only 4% of the population had detectable urinary TCC [8]. In another Canadian study, TCC was detected in less than 4% of infant urine and meconium samples and was not detected in infant formula or breast milk [9]. The authors attributed their findings, corroborated by the more recent study, to limited use of the chemical in Canada [9]. Similarly, a study 2 Journal of Toxicology done in Greece found a detection rate of 4% in urine with a mean level of 0.6 ng/mL (LDQ-1.9 ng/mL) [10]. In a 2013 Danish study done in mother-child pairs, TCC was detected at levels over the LOD in 25% of mothers and 28% of children, with mean TCC levels of 0.08 (LOD-1.3) and 0.07 (LOD-1.0) [11]. In a 2014 study of Danish pregnant women, TCC was found in 54% of samples tested [12]. When measured in children/adolescents, young men, and pregnant women, the compound was detected in 54%, 17%, and 18% of samples, respectively [13]. A study in a Chinese population found TCC in almost all (99-100%) of the urine samples tested, with high concentrations detected in the toenails and fingernails of the subjects [14]. Clearly, exposure to TCC varies by populations, with location, sex, age, and race/ethnicity possibly playing a role. This is likely due to the variable use of the chemical in consumer products by countries and individuals.
There has been very little research on the indirect exposure of offspring during development. However, in two studies by Enright et al., TCC has been shown to transfer to the fetus via maternal exposure in both human placental in vitro and rodent in vivo models [15,16]. In the in vivo models, TCC was found in maternal and fetal placental tissue, as well as in the fetus. TCC also transfers to offspring during lactation in exposed mothers [15,16].
TCC is an environmental contaminant (it was one of the most commonly detected organic wastewater and surface water contaminants in the US in 2011), with concentrations as high as 6.75 g/L. It is toxic to aquatic organisms (perhaps more so than triclosan), with an LC50 (i.e., the concentration of 50% lethality) as low as 3.1 g/L in certain organisms [17]. It has been shown to bioaccumulate in aquatic organisms, such as snails [17,18]. In fish, TCC also bioaccumulates. However, TCC has been shown to be rapidly metabolized and eliminated after absorption in fish, facilitated by the in vivo conversion of TCC to its sulfate and glucuronic acid conjugate metabolites (i.e., 2 -O-Gluc-TCC, 2 -O-SO3-TCC, 2 -OH-TCC, 3 -OH-TCC, and 6-OH-TCC). Thus, the bioaccumulation in fish is less than would be predicted by the n-octanol/water partition coefficient of TCC [19].
The route of metabolism and the metabolites produced are similar in humans and other mammalian species [20][21][22][23][24]. In humans exposed orally to TCC, the urinary elimination half-life was about 20 hours [21]. With intravenous injection, the urinary elimination half-life was estimated to be 10 hours. In the same study, individuals were exposed to TCC through a whole body wash in the shower. The urinary elimination halflife in this case was 28 hours; however, the bulk of excretion was detected in the feces, which was detected 12-20 days after exposure [25]. The fact that some human populations had up to 100% detection indicates the ubiquitous exposure of TCC in certain groups [14].
Although TCC has been widely used for over 50 years, it was only recently that concerns were raised about its endocrine disruptive properties. In 2016, the FDA banned its use in over-the-counter hand and body washes [26]. This was due to the fact that manufactures were unable to show that TCC was safe for daily use or was any more effective than regular soap alone. However, TCC is still used in other countries (as evidenced by human exposure) [11,12,14] and the chemical is still approved in the US for all other personal care products, including deodorants, lotions, and toothpaste, as well as for medical and healthcare applications [26]. A 2007 study put forth the idea that TCC was a "new kind of endocrine disruptor" that augmented the action of endogenous hormones rather than directly activating hormone receptors [27]. More recently, TCC has been shown to disrupt the gut microbiome in animals [28] and humans [29], which, in turn, can have myriad effects on health [30]. Because of these potential endocrine-disruptive properties of TCC and the potential for TCC to be transferred to offspring during development, we conducted a scoping review to catalogue and map the literature-to-date on the endocrine, reproductive, and developmental effects of TCC.

Literature Search and Study Identification.
A comprehensive literature search was performed to identify studies describing the endocrine, reproductive, and developmental effects of exposure to TCC. The search included all articles published for all years to August 2017. Electronic searches were performed in Web of Science, PubMed, and TOXLINE using the following search criteria: triclocarban OR trichlocarban OR triclocarbanum OR trilocarban OR (N-(4-chlorophenyl-N-(3,4-dichlorophenyl)) urea) OR (N-(3,4-dichlorophenyl-N-(4-chlorophenyl) urea) OR (1-(3 ,4 -Dichlorophenyl) -3-(4 -chlorophenyl)) urea) OR trichlorocarbanilide OR trichlorodiphenylurea OR 101-20-2. 101-20-2 is the Chemical Abstracts Services (CAS) Registry Number for triclocarban. For inclusion, the studies had to be original works, be in the English language, and assess endocrine, reproductive, or developmental effects of triclocarban exposure, including human, in vitro, and in vivo models. All titles and abstracts were screened for inclusion independently by two reviewers using the software DistillerSR5 (Evidence Partners, Ottawa, Ontario, Canada). Conflicts or discrepancies were resolved by the two reviewers.

Data Extraction.
Parameters collected during data extraction included author information, year of publication, summary of endpoint(s) evaluated, information about the model used (e.g., cell line and species), concentrations/doses tested, exposure duration, age at exposure, and route of exposure. The data extraction entries were inputted by one reviewer and then quality-checked by a second reviewer. Any discrepancies were resolved by discussion.

Results
Our searches for potentially relevant studies resulted in 1560 articles. A total of 32 studies (2 human, 19 in vivo animal, and 11 in vitro only) were identified as relevant and these studies underwent full review and data extraction. The summary statistics of the body of literature are presented in Table 1. The majority of the research on this topic has been published in the years since 2010, with over 90% of the literature published in the past 7 years. Studies were identified in all three categories: development, endocrine, and reproduction. Doses in aquatic studies ranged from 100 ng/L to 10000 g/L, with many studies in the range of the 6.75 g/L found in US surface waters. The mammalian studies generally exposed animals to high doses, although a recent 2017 study used 100 nM, with the authors indicating that this was an "environmentally relevant concentration" [16].

In Vitro/Steroid Activity.
In systematic review frameworks, such as the Office of Health Assessment and Translation (OHAT) Systematic Review Framework, in vitro mechanistic evidence can be quantitatively used to support health hazard conclusions [50]. The in vitro studies are presented in Table 4. Fourteen studies assessed the steroidal activity of TCC, which may support the biological plausibility of the reproductive/endocrine effects of TCC in vivo [27,41,42,[51][52][53][54][55][56][57][58][59][60][61]. These studies measured steroid and steroid receptor activity, including estrogen, androgen, progesterone, thyroid, and glucocorticoid, as well as CAR activity. Table 5 shows the studies organized by the specific steroidal activity assessed. Some of these studies tested the possible steroid-enhancing activity of TCC, while others looked at the direct activities of TCC alone.

Discussion
Scoping reviews systematically map areas of research and can have several applications. They are employed during the first step of a systematic review in order to identify sources and types of available research to determine the feasibility/utility of carrying out a systematic review and to prioritize endpoints that can be evaluated through systematic review. They can also be used to help identify data gaps and direct future research. They have been used in the clinical sciences for many years [62][63][64] and have more recently been employed as tools in environmental health [65,66]. While scoping reviews are extremely useful for generating hypotheses for systematic review, they do not themselves answer research questions (e.g., whether or not a chemical is harmful to human or environmental health) [67][68][69][70]. In this scoping review, we aimed to identify and map the literature-to-date of the endocrine, developmental, and reproductive effects of TCC on organisms in order to identify endpoints for future systematic review and determine if additional research is needed in specific areas.

Possible Systematic Review Endpoints.
Our recommendations for future systematic reviews are based on several variables. In particular, endpoints suitable for systematic review ideally have literature in all three "evidence streams" (human, animal, and in vitro), although literature in all streams is not required for final analyses. Meta-analysis, which usually requires at least three studies on a specific endpoint, is an optional component of systematic reviews, and is especially useful when there are "conflicting" results from studies. Meta-analyses are helpful for determining the degree of hazard, if any, which is posed to humans [50]. The characteristics of the recommended systematic review endpoints are presented in Table 6. When examining reproductive/endocrine endpoints, there is sufficient literature on the estrogenic and/or androgenic effects of TCC in animals to undertake a systematic review, including three rodent studies and eight nonmammalian studies. Further, there are many in vitro studies that might support the biological plausibility of the estrogenic/androgenic action of TCC, as most of the in vitro studies assessed these endpoints (Table 5). Although there are no human studies assessing these endpoints, there are sufficient studies (5) to evaluate vitellogenin expression in a meta-analysis, if desired. Vitellogenin is an estrogenresponsive egg protein in egg-laying animals and, when found in males, indicates exposure to estrogenic chemicals [71]. Both the animal and in vitro literatures in this area have studies examining the direct effects of TCC as well as steroid augmentation effects. A review employing systematic methodologies, comparing these two types of effects, could shed light on the mechanisms of TCC activity. There are fewer studies on specific developmental endpoints, and therefore the developmental effects of TCC may not yet be mature enough for conducting a systematic review. One possible endpoint to pursue, however, is offspring size (birthweight and lifetime weight). This endpoint is represented in 1 human study [32] and 6 animal studies (2 rodent and 4 nonmammalian) [16,33,35,37,39,40]. The fact that all three data streams are represented is a positive aspect for systematic review. There is also mechanistic evidence that could possibly be used to support the biological plausibility of TCC's effects on offspring growth, for example, TCC's effects on metabolic genes in vivo [16] and TCC activity on the thyroid receptor in vitro [55]. In all, however, there is little research on specific developmental endpoints.

Future Research.
In order to protect human and wildlife health as quickly and efficiently as possible, further research of the effects of TCC should be prioritized. Overall, studies designed to be relevant to human health and risk assessment are needed, including identifying sensitive target tissues and functional assays, employing doses within the range of human exposure and using relevant models. Table 7 outlines the state of the current research and identifies data gaps with respect to model and endpoint. While mechanistic models seem to be well represented, human and mammalian models are less so.
There are also data gaps with respect to endpoint. One endpoint that appears to be particularly responsive to low exposures (i.e., comparable to those reported in humans) is increased weight in offspring following developmental exposure [16]. This should be further explored in developmental animal studies and prospective epidemiological studies that follow the growth of children. In vitro studies exploring possible mechanisms for these actions would add to the body of literature as well. In general, more human studies would be helpful in understanding the effects of TCC, as we only found two human studies documenting the effects of TCC exposure.
For endocrine/reproductive effects, the majority of the literature is in aquatic species. Endpoints in these papers, such as vitellogenin induction, appear to be sensitive to low doses of TCC. However, the current endocrine/reproductive literature in mammals is less robust, and the endpoints studied, such as reproductive organ weights, are not as sensitive. Further research in this area should include mammalian models, with endpoints that are responsive to low doses of chemicals, such as ovarian histology and mammary disruption.
Lastly, the finding that TCC acts as a "steroid augmenter" should be studied further. The ability of endocrine disruptors to enhance the activity of endogenous hormones has not been extensively explored. TCC (and triclosan) appears to act this way in vivo and in vitro, and this may be a unique characteristic of these chemicals. Further study of this mechanism (and inclusion of assays that identify this activity among more chemicals) could be informative to the broader field of endocrine disruption.

Conclusions
Our scoping review found that systematic review(s) of the effects of TCC on human health could be carried out using Journal of Toxicology 5 .0 g/L 0.14 g/L After 4 weeks (but not before), TCC-exposed snails had significantly increased unshelled, shelled, and total embryos (there were some nonmonotonic results with shelled and total embryos).

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Journal of Toxicology There was no effect on implantation number, maternal organ weights, or hormone profile (E2, P, T, T4, and TSH). Exposure to pups from gestation through lactation did not affect number of pups born or birth weight. However, pups exposed to TCC from gestation through lactation did not survive past PND8 and there was evidence of mammary gland involution in the dams. Body weight and survival were decreased in pups nursed by TCC exposed dams. Schultz  1 g/L Fecundity was decreased by greater than 50% in fathead minnows exposed to 5 g/L TCC but not in those exposed to 1 g/L. Fecundity was further decreased with coexposure to 5 g/L TCC and 0.5 g/L 17b-trenbolone. Chronic exposure to TCC for 22 d did not affect GSI or male secondary sex characteristics and did not cause masculinization of female fish. This exposure also did not alter plasma VTG concentrations or circulating E2 or T concentrations. In contrast, ex vivo assessment of steroid production from the reproductive organs was altered by TCC. Fish exposed to 5 g/L TCC had more preovulatory atretic follicles and other abnormalities. Gene expression changes in the ovary were also observed in fish exposed to 5 g/L TCC. CYP19a was decreased in the testes and disrupted in the ovaries of exposed fish. Liver VTG expression was also altered in males. Yueh   0.2, 0.5% w/w TCC 0.2% w/w TCC TCC exposure to pups from gestation through lactation did not affect number of pups born or birth weight. However, pups exposed to TCC from gestation through lactation did not survive past PND8. Body weight and survival were decreased in pups nursed by TCC-exposed dams. 13 g/L On a population level, TCC reduced population density of daphnia immediately following exposure. Mortality was size-and age-dependent with neonates being more sensitive than adults. Mortality was decreased when multiwalled carbon nanotubules were present in the growth medium. Exposure to TCC or TCC + multiwalled carbon nanotubules increased the ratio of juveniles to adults in the population but had no effect on minimal or maximal body length.

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Journal of Toxicology         NA NA 0.13 g/L (cord blood plasma); 3.44 g/g creatinine (urine) Rodent 0.2-0.5% w/w 0.25% w/w 100 mM, 0.2-0.5% w/w TCC Aquatic 0.045-14.0 g/L 0.55-10 g/L 0.1-10000 g/L In vitro 1 × 10 −9 -1 × 10 −3 M 1 × 10 −9 -2 × 10 −3 M 3 × 10 −6 -3 × 10 −2 M * vitro/in vivo assessments counted separately, even if included in the same publication. * * Thyroid activity. the current literature, using a framework such as the OHAT Systematic Review Framework [50] or the Navigation Guide [69,70]. The best candidate endpoints are the estrogenic and/or androgenic effects and offspring birth weight/growth. However, future research could add to the body of literature of each of these endpoints and improve the confidence of the results of any systematic reviews carried out. In particular, there are very few mammalian studies and almost no human studies. Thus, future research should include animal and human developmental growth studies and mammalian studies that include sensitive endocrine/reproductive endpoints. Systematic reviews of these topics, especially with the goal of deriving human health hazard conclusions of TCC effects, could aid in future research and regulation in order to most effectively protect human health.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Foundation, Wallace Genetic Foundation, and the International Chemical Secretariat (ChemSec). The authors would like to thank Christina Ribbens for help in literature procurement and cataloguing.