Maternal Reproductive Toxicity of Some Essential Oils and Their Constituents

Even though several plants can improve the female reproductive function, the use of herbs, herbal preparations, or essential oils during pregnancy is questionable. This review is focused on the effects of some essential oils and their constituents on the female reproductive system during pregnancy and on the development of the fetus. The major concerns include causing abortion, reproductive hormone modulation, maternal toxicity, teratogenicity, and embryo-fetotoxicity. This work summarizes the important studies on the reproductive effects of essential oil constituents anethole, apiole, citral, camphor, thymoquinone, trans-sabinyl acetate, methyl salicylate, thujone, pulegone, β-elemene, β-eudesmol, and costus lactone, among others.


Introduction
The female reproductive cycle involves a very complex sequence of changes in the uterus, ovaries, breasts, and regulatory hormone levels. Several mechanisms, metabolic pathways, and enzymes are involved in controlling and regulating reproductive hormone levels in the blood. During the reproductive cycle, these endogenous hormones are responsible for preparing for implantation and for milk production [1,2]. Out of concern of adversely affecting the unborn child, some pregnant women prefer to use herbs, herbal preparations, or oils rather than conventional medication to treat pregnancy-related symptoms (morning sickness, nausea, vomiting, heartburn, etc.) [3,4]. Indeed, several plants can improve the female reproductive function and some are beneficial during pregnancy, childbirth, and postpartum [5,6]. Similarly, essential oils (EOs) are generally safe, and many oils have a generally recognized as safe (GRAS) status. However, the use of herbs and EOs during pregnancy is a highly controversial matter. It is worth mentioning that it is the individual composition of an EO and the possible hazardousness of a single or a group of constituents that determine their medical and therapeutic usage. Some EO-containing plant species are highly variable and may produce several EO chemotypes with different EO compositions of which one or some chemotypes possess potential maternal reproductive toxicity. Causing abortion is a major concern. It is generally believed that EOs extracted from emmenagogic plants are dangerous or unsafe in pregnancy, as they might cause menstrual bleeding and lead to miscarriage. Yet, that is not always true. The oils do not necessarily carry the same activity as the whole plant. Regardless of their ability to promote menstruation, there is no decisive evidence that these oils are abortifacient in aromatherapy amounts. For instance, the whole plants of savin, pennyroyal, tansy, and rue can induce miscarriage and their oils were on the list of abortifacient oils at some point [7]. Thus far, these oils showed no activity on uterine muscle of isolated human uterus [8] and did not cause fetal death [9,10]. Still, these facts do not prove the safety of these oils. Since the   (52.5-84.3%), fenchone (4.0-24.0%), α-pinene (tr-10.4%), d-limonene (0.5-9.4%), and estragole (2.8-6.5%).
(E)-Anethole was reported to have anti-hypernociceptive, anticancer, antiplatelet, anti-inflammatory, and anesthetic properties [75][76][77]. However, administration of (E)anethole-rich EOs (by any route) should be avoided in pregnancy, breastfeeding, and estrogen-dependent cancers. Additionally, internal use of (E)-anethole-rich EOs is not advisable in childbirth due to its antiplatelet aggregation activity. There is enough evidence for the estrogenic action of (E)-anethole. (E)-Anethole was estrogenic in yeast assays (in vitro) [1,78]. It was reported to bind to estrogen receptors in engineered yeast cells [1]. In humans, sweet fennel tea (rich in (E)-anethole) was estrogenic in vivo [79]. A notable increase in uterine weight was observed in immature female rats following (E)-anethole treatment (80 mg/kg/day for 3 days), confirming its estrogenic effect [80]. (E)-Anethole showed an anti-implantation effect in pregnant rats. Oral administration of (E)-anethole (50, 70, and 80 mg/kg on gestational days 1-10) to pregnant albino Charles Foster rats caused a dose-dependent reduction in implantation as a result of a disruption of hormonal balance [80]. It is worth mentioning that both mice and humans can metabolize (E)-anethole in a similar way, while rats metabolize it differently [81]. A metabolite of anethole, anethole-1',2'-epoxide, was carcinogenic and caused the formation of hepatomas and papillomas in mice [82].
Anethole-rich essential oils such as aniseed, star anise, bitter fennel, sweet fennel, and aniseed myrtle are estrogenic in one or more in vitro assays and may cause reproductive hormone modulation [1,78]. These oils are hepatotoxic due to their high (E)-anethole content. Bitter fennel oil is hepatotoxic due to the metabolite, anethole-1',2'-epoxide [83]. Like bitter fennel EO, sweet fennel EO is a reproductive hormonal level modulator, fetotoxic, and hepatotoxic [84]. Sweet fennel tea (containing 1.3-10.0% of the oil [85]) showed in vivo estrogenic activity in humans and its prolonged use caused premature breast development and significantly higher serum estradiol levels [79]. A sweet fennel oil (with 72% (E)anethole, 12.0% fenchone, and 5% estragole) was teratogenic at 0.93 mg/mL and produced about 50% reduction in differentiated rat embryo limb bud foci. It dose-dependently decreased the intensity of oxytocin or prostaglandin E2-induced uterine contractions ex vivo [86] which is why the use of sweet fennel oil is not advisable during slow-progressing labor. Therefore, consumption of anethole-rich essential oils is unsafe and should be avoided (by any route) during pregnancy, breastfeeding, and in some estrogen-dependent cancers [87,88]. These oils are potentially carcinogenic based on their estragole and safrole (minor components) content [89]. (E)-Anethole and estragole interfered with fetoplacental steroidogenesis in a co-culture of human adrenocortical carcinoma cells (H295R) and human placental choriocarcinoma cells (BeWo) cells by increasing hormonal concentrations and altering steroidogenic enzyme activity and expression [90,91].

Methyl Salicylate-Rich Essential Oils
Methyl salicylate is a phenolic ester that dominates wintergreen (Gaultheria procumbens L.) (96.0-99.5%) [92] and sweet birch (Betula lenta L.) (90.4%) oils [22]. Methyl salicylate is largely hydrolyzed into salicylic acid in the liver [93]. Following topical application in humans, methyl salicylate can be transdermally absorbed and converted to salicylic acid in the dermal and subcutaneous tissues [94,95]. Orally taken methyl salicylate is metabolized faster in rats and dogs than in humans which means a higher toxicity in humans [96]. Methyl salicylate poisoning in humans is known to cause fever, nausea, vom-iting, CNS excitation, tachycardia, rapid breathing, high blood pressure, respiratory failure, pneumonia, pulmonary edema, convulsions, and coma [97]. Methyl salicylate poisoning in humans has a 50-60% mortality rate which is a result of cardiovascular collapse and respiratory failure [98,99]. Methyl salicylate showed in vitro human estrogen receptor α (hERα) agonistic activity [100]. Salicylates have been shown to cross the placenta [101] and lead to restricted growth and congenital abnormalities in animal experiments [102]. The fact that superoxide dismutase treatment prevented salicylate-induced malformations in rat embryos suggests that free oxygen radicals play a role in its teratogenic action [103]. Intraperitoneal injection of methyl salicylate (200 or 400 mg/kg, pregnant rats on gestational days 9 and 10) dose-dependently reduced the development of the brain, lung, liver, and kidney of the fetus [104]. Methyl salicylate (i.p., 50 or 100 µL) given to female rats on gestational days 10 and 11 resulted in retardation of fetal kidney development. At 100 µL, maternal weight gain was retarded, the offspring were fewer and smaller, and resorptions and malformations were increased [105]. A single subcutaneous injection of methyl salicylate (1.5 mL/kg on gestation day 7, 9, or 11) to female rats resulted in higher fetal deaths, reduced fetal weight, and cleft palate and tail abnormalities [106]. At 200, 250, or 300 mg/kg/day (i.p., on gestation days 11-12), methyl salicylate showed teratogenicity and embryotoxicity in pregnant Sprague-Dawley rats [107]. Methyl salicylate increases the occurrence of dilated renal pelvis in the rat fetus and causes a temporary maturation delay in the rat's ability to concentrate urine [107]. In another experiment, several anomalies were observed in the offspring digestive tract, CNS, liver, and skeleton following a single subcutaneous injection of methyl salicylate (0.1-0.5 mL on gestational day 9, 10, or 11) in pregnant rats [108]. Subcutaneous administration of methyl salicylate (400 mg/kg) caused a substantial decrease in plasma calcium levels in pregnant rats and mice which might be linked to the fetal toxicity [109]. In pregnant hamsters, oral or topical methyl salicylate caused neural tube fusion failure in the embryos [93,110]. In addition, high oral doses of wintergreen EO were toxic and teratogenic in rats and monkeys [102]. Based on the available information, use of methyl salicylate-rich essential oils or any preparations containing them, by any route, should be avoided during pregnancy and lactation.
Another sabinyl acetate-rich oil is savin oil (Juniperus sabina L.). Savin oil (50% sabinyl acetate) is embryo-fetotoxic, abortifacient, and hepatotoxic [116]. It can easily cross the placenta and cause abortion [117]. Subcutaneous administration of savin EO to pregnant mice (at 15, 45, or 135 mg/kg on gestational days 6-15) caused embryotoxicity and sig-nificant weight loss [116]. It also inhibited implantation in mice on gestational days 0-4 but not on gestational days 8-11 suggesting that sabinyl acetate causes abortion [111]. The abortifacient action of the savin plant does not seem to be only due to the oil. An ether extract of Juniperus sabina, prepared after isolating the oil, showed anti-implantation effect in a dose-dependent manner [118]. Since nothing much can be gained from using savin oil, it should not be used either internally or externally. Similarly, juniper berry (Juniperus communis L.) ethanolic extract was clearly abortifacient [119]; however, there is no evidence that the juniper berry EO is abortifacient.
Moreover, Spanish sage oil is a well-known abortifacient. Subcutaneously injected Spanish sage oil fraction (50% sabinyl acetate) into pregnant mice (at 15, 45, and 135 mg/kg on gestational days 6-15) caused abortion and maternal toxicity in a dose-dependent manner [115]. Spanish sage oil (0.01 mg/mL) also induced β-galactosidase activity in yeast which suggests a possible estrogenic activity [120]. Similarly, wormwood oil is neurotoxic, embryo-fetotoxic, and abortifacient [23,39]. It is particularly hazardous since it carries combined risks from thujones and sabinyl acetate. Since there is no established no observed adverse effect level (NOAEL), it is best to completely avoid sabinyl acetate-rich essential oils and any preparations containing them in pregnancy, especially during the first trimester.

Apiole-Rich Essential Oils
For many years, parsley (Petroselinum crispum (Mill.) Fuss) and its concentrated preparations have been used in South America and Italy to induce abortion, which often ended in death due to severe post abortive vaginal bleeding [136]. The abortifacient effect is attributed to parsley apiole, a main component in most parsley leaf and seed oils. Parsley apiole and dill apiole are bicyclic phenylpropenoid ethers. Parsley apiole is found in parsley seed oil (11.3-67.5%) [58] while dill apiole is found in Indian dill (Anethum sowa Roxb. ex Flem.) seed oil (20.7-52.5%) [23] and parsley leaf oil (0.2-5.2%) [22,57]. Parsley apiole poisoning causes severe neurotoxicity which presents a risk of abortion [137]. Signs of parsley apiole intoxication include fever, severe abdominal pain, vaginal bleeding, abortion, convulsions, vomiting, and diarrhea [138]. A single gavage dose of parsley apiole (10 mL/kg) was sufficient to kill all experimental mice within 60 hours due to liver and kidney toxicity [139]. Doses of 5-14 g caused severe hemorrhage and induced abortion in pregnant rabbits [140]. Parsley leaf and seed oils are hepatotoxic, nephrotoxic, and may be abortifacient if taken orally. Topical application of parsley oils is also inadvisable during pregnancy [88]. Since there are no safety thresholds for parsley apiole in humans, internal and external use of parsley apiole-rich essential oils is not recommended in pregnancy due to the high risk of abortion. The structural similarity to parsley apiole suggests that dill apiole could carry the same toxicity and could be hazardous in pregnancy. Therefore, it is best to avoid apiole-rich oils (all routes) throughout pregnancy and breastfeeding [22].

Camphor-Rich Essential Oils
Camphor is a common component in many essential oils. It is a major component of Ho (Cinnamomum camphora (L.) J.Presl) leaf oil (camphor chemotype) (37.8-84.1%) [40], feverfew (Tanacetum parthenium (L.) Sch.Bip.) oil (28.0-44.2%) [37], and Spanish lavender (Lavandula stoechas L.) oil [64]. Upon consumption, camphor is absorbed immediately via the mucosa and can freely cross the placenta in pregnant women [98] and reach the fetal organs such as brain, liver, lungs, and kidneys [141]. At very high doses, camphor can cause hemorrhage due to severe damage to the placenta [142]. In female mice, camphor (300 mg/kg/day for 20 days) increased the activities of hepatic CYP, glutathione S-transferase, and aryl hydrocarbon hydroxylase [143]. Camphor is very toxic to humans with a lethal dose of 5-20 g [144] and 50-550 mg/kg [98]. Camphor can cause damage to several organs including liver, kidney, and brain [145,146]. It can also cause convulsions [147] and induce seizures [148]. Signs of camphor poisoning include seizures, lack of coordination, respiratory depression, nausea, vomiting, and coma [125,149,150]. Despite being neurotoxic, hepatotoxic, and lethal in high doses, camphor is unexpectedly non-teratogenic and non-embryotoxic. In almost fatal doses, it can be reproductively toxic and abortifacient because the fetus lacks the necessary enzymes to metabolize it [151]. Camphor caused a dose-dependent maternal toxicity when given orally to pregnant rats (0.216, 0.464, or 1 g/kg/day on gestational days 6-17) and pregnant rabbits (0.147, 0.316, or 0.681 g/kg/day on gestational days 6-18) [152]. Yet, since camphor is believed to be more toxic to humans than animals, camphor-rich oils should be avoided in pregnancy and breastfeeding.
Citral is not mutagenic or carcinogenic [155]. However, it has shown some reproductive toxicity in animal studies. Citral reduced the fertility of female Wistar rats through decreasing the number of normal ovarian follicles [156]. Citral is a well-known retinoic acid synthesis inhibitor [157][158][159][160]. In epithelial tissues, citral has been shown to antagonize the activity of vitamin A and prevent the oxidation of retinol to retinoic acid [161]. In mouse epidermis, citral inhibited tissue morphogenesis and tumor production [159,162]. When tested on embryos of white Leghorn chicken, citral showed a dose-dependent teratogenic effect represented by inducing malformations and abnormal eye development [163][164][165]. Citral was reported to act via suppressing the activity of the enzyme ALDH1A1 responsible for retinoic acid synthesis, which in turn affects fetal development [166]. Citral (55 mM) partially inhibited the initiation of meiotic division in human fetal ovary tissues which relies partially on retinoic acid [166]. The oral NOAEL for citral-induced prenatal toxicity was set as <60 mg/kg/day [167]. Orally administered citral (at 60, 125, 250, 500, and 1000 mg/kg on days 6 to 15 of pregnancy) produced signs of embryo-fetotoxicity (growth retardation, skeletal abnormalities, and spleen weight increase) and maternal toxicity (decreased maternal weight gain, increased resorptions, and impaired implantation) in pregnant Wistar rats [167]. Citral was teratogenic in studies with chick embryos [168,169]. The teratogenic effects of citral were also observed in Xenopus embryos treated with 60 mM [170]. After exposure to 1.75 mM of citral for 24 h, tooth development was completely inhibited in 70% CD-1 Swiss mouse embryonic mandible explants while the addition of retinoic acid restored odontogenesis [171]. When injected intra-abdominally into pregnant BALB/c mice (>35 mmol/g on the 9th gestational day), citral caused fetal cranial chondrogenesis and osteogenesis restrictions that diminished by adulthood [158]. However, when given to pregnant Wistar rats (by gavage at 125, 250, 500, or 1000 mg/kg on gestational days 6-15), citral caused maternal toxicity, a dose-dependent increase in resorptions per implantation, and a slight teratogenicity [167]. The mechanism of action seems to involve competing with estrogen for estrogen receptors [172]. When applied directly to the rat's vagina, citral showed estrogenic effects and caused vaginal hyperplasia [172]. Inhalation of citral (for 6 hr/day on gestation days 6-15 at 10 or 34 ppm as vapor, or 68 ppm as an aerosol/vapor mixture) did not cause teratogenicity in Sprague-Dawley rats [168]. Due to their high citral content, Australian lemon balm, honey myrtle, lemon basil, lemon petitgrain, lemon myrtle, lemon thyme, lemongrass, lemon tea tree, May chang, Melissa, and lemon verbena EOs are teratogenic and their internal use should be restricted during pregnancy [153].
For a long time, pennyroyal has been used as an abortifacient even with its potentially deadly hepatotoxic effects [183]. Pennyroyal oil is hepatotoxic and neurotoxic due to the high content of (6R)-(+)-menthofuran and (1R)-(+)-β-pulegone [179]. Both Mentha pulegium oil and pulegone prevented rat uterine muscle contraction [8]. Pennyroyal intoxication causes severe liver damage, internal hemorrhage, and pulmonary edema [184,185]. Intraperitoneal administration of both pennyroyal oil and pulegone showed similar effects in mice [179]. Since there is no significant medicinal benefit from using β-pulegone-rich oils and due to their hepatotoxicity and the potential of causing abortion, it is best to avoid them in pregnancy and breastfeeding [186].

Thymoquinone-Rich Essential Oils
Thymoquinone is a bicyclic benzenoid ketone found in black seed (Nigella sativa L.) oil (26.8-54.8%) [27]. It showed reproductive toxicity with an i.p. NOAEL of 15 mg/kg. When administered daily to rats (i.p., at 8 mg/kg), thymoquinone killed most of the animals within a week and the surviving animals had severe peritonitis [190]. It suppressed VEGF-induced angiogenesis in the matrigel plug assay. Subcutaneous administration of thymoquinone to mice (at 6 mg/kg for 15 days) abolished angiogenesis in prostate cancer tumors [191]. Administration of a single dose of thymoquinone (i.p., at 35 or 50 mg/kg on gestational days 11 or 14) to pregnant rats caused a dose-dependent fetal resorption and maternal toxicity [192]. Thymoquinone (by gavage at 10 mg/kg/day on gestational days 1-19) reduced malondialdehyde formation and increased hepatic glutathione in mice with induced gestational diabetes [193]. Due to its strong anti-angiogenic activity [191] and reproductive toxicity, thymoquinone is mostly hazardous in pregnancy. Black seed oil may be fetotoxic because of its high thymoquinone content [191]; therefore, its consumption during pregnancy and breastfeeding should be avoided.

Other Essential Oils
(E)-Cinnamaldehyde-rich oils, such as cassia (Cinnamomum cassia (L.) J. Presl) [23] and cinnamon (Cinnamomum verum J. Presl) bark oil [23,24], carry a risk of embryotoxicity and should be avoided during pregnancy and breastfeeding. Cinnamon bark oil has GRAS status, yet it has been shown to lower the number of nuclei and affect the distribution of embryos in pregnant mice (orally, at 375 mg/kg for 2 weeks) [202].
Some oil chemotypes like the estragole chemotype of basil (Ocimum basilicum L.) oil [23] are toxic based on their estragole content which, in high concentrations, is carcinogenic and should be restricted during pregnancy and lactation [88,89]. Dalmatian sage oil carries a combined risk from its camphor and thujone contents which makes it neurotoxic and embryotoxic [36]. The oil should not be taken orally and its consumption is contraindicated in pregnancy and breastfeeding [88]. Ingestion of dalmatian sage oil can cause convulsions, seizure, coma, and may lead to death [209,210]. Dalmatian sage oil (0.25%, 375 mg/kg for 2 weeks) negatively influenced the distribution of embryos according to nucleus number when fed to pregnant mice [202]. Since the risks of dalmatian sage oil outweigh its benefits, it is best to avoid using it. Hibawood (Thujopsis dolabrata (L.f.) Siebold and Zucc.) EO may present a reproductive toxicity because of its β-thujaplicin content [22]. In rats, orally delivered β-thujaplicin caused fetal malformations at 135 mg/kg and a decrease in fetal weight at 45 mg/kg [211]. Nasturtium (Tropaeolum majus L.) flower absolute carries a moderate toxicity because of its benzyl cyanide content [22].
In some cases, the hazardous components in the oil have not been identified yet. For instance, carrot (Daucus carota L.) seed oil has GRAS status; however, it may interfere with gestation and should be avoided altogether during pregnancy and breastfeeding. It is worth mentioning that the wild carrot plant is reputed as a contraceptive agent. Dong and colleagues have reported that carrot seed oil caused antigestational effects in rats and mice [212]. Subcutaneous injection of carrot seed EO (2.5-5 mL/kg) to female rats and mice inhibited implantation and prevented progesterone synthesis [213]. Another example is oregano (Origanum vulgare L.) oil [53][54][55][56]. Although oregano oil has a GRAS status, it is embryotoxic. Orally delivered Origanum vulgare EO to pregnant mice (about 150 mg/kg for two weeks) caused an increase in the rate of embryonic cell death [202]. Additionally, zedoary (Curcuma zedoaria (Christm.) Roscoe) oil has a GRAS status but its consumption can interfere with gestation and can induce abortion [22]. There was obvious embryotoxicity for zedoary EO ex vivo and reproductive toxicity in animal and developmental experiments [14,174]. In addition, the oil was anti-angiogenic in mice [174], suggesting a strong link between its anti-angiogenic and embryotoxic effects [14]. Chinese zedoary EO (i.p., 300 mg/kg) prevented implantation in a dose-dependent manner in female rats on gestational days 7-9 and prevented about 77% of pregnancies. When administered intra-vaginally to female rabbits at 60 or 400 mg/kg/day on gestational days 5-9 and 2-4, a steam-distilled zedoary EO prevented 16% and 100% of pregnancies, respectively [22]. Aqueous extracts of C. zedoaria rhizome (10 g/kg/day for 20 days) demonstrated reproductive toxicity in pregnant mice [214]. The embryotoxic effect of zedoary EO was attributed to its sesquiterpenoids, which can block VEGF-mediated angiogenesis [14]. Nevertheless, there is no direct evidence to link any of the oil components to its antifertility effect. Moreover, zedoary rhizome decoctions and ethanol extracts also have antifertility effects [215].
Rue (Ruta graveolens L.) oil may be abortifacient and should be strictly prohibited in pregnancy and breastfeeding. In South America, Ruta graveolens ingestion caused abortion [136]. Ingestion of rue aqueous extract was abortifacient, and inhibited implantation in rats at 1 mL/kg [9]. Rue chloroform extract showed antifertility effects due to the presence of chalepensin [9]. Pilocarpine, a compound not present in rue oil, has been suggested as the abortifacient agent [216]. Very little information was found about the toxicity and safety of rue oil. Until further data are available, rue oil should be avoided altogether during pregnancy.
In summary, if essential oil constituents are present in the mother's circulation, they are expected to reach the fetus and exert some toxic effects. Due to the lack of clinical evidence on reproductive toxicity in humans, it is best to avoid or restrict the use of potentially dangerous essential oil constituents such as anethole, apiole, citral, camphor, thymoquinone, trans-sabinyl acetate, methyl salicylate, thujone, pulegone, β-elemene, β-eudesmol, and costus lactone.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
EO essential oil CNS central nervous system GSK-3β glycogen synthase kinase-3β sc subcutaneous PMS premenstrual syndrome GRAS generally recognized as safe i.p. intraperitoneal IC 50 median inhibitory concentration NOAEL no observed adverse effect level ppm parts per million VEGF vascular endothelial growth factor GABA A gamma-aminobutyric acid type A