Toxicological evaluation of isopropylparaben and isobutylparaben mixture in Sprague–Dawley rats following 28 days of dermal exposure

Regulatory Toxicology and Pharmacology xxx (2015) 1e8

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Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure Min Ji Kim a, Seung Jun Kwack b, Seong Kwang Lim a, Yeon Joo Kim a, Tae Hyun Roh a, Seul Min Choi a, Hyung Sik Kim a, *, Byung Mu Lee a, ** a

Division of Toxicology, School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, South Korea Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University, 92 Toechonro, Uichang-gu, Changwon, Gyeongnam 641-773, South Korea

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 December 2014 Received in revised form 28 July 2015 Accepted 30 August 2015 Available online xxx

The alkyl esters of p-hydroxybenzoic acid (Parabens) have been of concern due to their probable endocrine disrupting property especially in baby consumer products. The safety of parabens for use as a preservative in cosmetics has come into controversy, and thus consumer demand for paraben-free products is ever increasing. Thus, more comprehensive studies are needed to conclusively determine the safety of the multiple prolonged exposure to parabens with cosmetic ingredients. This study was conducted to investigate the potential repeated 28 days dermal toxicity (50, 100, 300, or 600 mg/kg bw/ day) of isopropylparaben (IPP), isobutylparaben (IBP), or the mixture of IPP and IBP in rats. There were no significant changes in body and organ weights in any group. However, histopathological examinations showed that weak or moderate skin damages were observed in female rats by macroscopic and microscopic evaluations. In female rats, no observed adverse effect levels (NOAELs) of IPP with no skin lesion and IBP for skin hyperkeratosis, were estimated to be 600 mg/kg bw/day, and 50 mg/kg bw/day, respectively. With regard skin hyperkeratosis, the lowest observed adverse effect level (LOAEL) of the mixture of IPP and IBP was estimated to be 50 mg/kg bw/day. Analysis of six serum hormones (estrogen, testosterone, insulin, T3, TSH, or FSH) in animals showed that only FSH was dose-dependently decreased in the mixture groups of 100 mg/kg bw/day or higher. These data suggest that the mixture of IPP and IBP showed a synergistic dermal toxicity in rats and should be considered for future use in consumer products. © 2015 Elsevier Inc. All rights reserved.

Keywords: Isopropylparaben Isobutylparaben Cosmetics Mixture toxicity NOAEL

1. Introduction Parabens are general name of all kinds of alkyl ester of phydroxybenzoates. They have been used as preservatives, stabilizers and antiseptics, due to their ability to change conformational structure of enzyme of germs which inhibit germs to utilize dihydrofolate (Satoh et al., 2000; Tzortzatou and Hayhoe, 1974). They

* Corresponding author. Division of Toxicology, School of Pharmacy, Sungkyunkwan University, Cheoncheon-Dong 300, Changan-Ku, Gyeonggi-Do, Suwon, 440-746, South Korea. ** Corresponding author. Division of Toxicology, School of Pharmacy, Sungkyunkwan University, Cheoncheon-Dong 300, Changan-Ku, Gyeonggi-Do, Suwon, 440-746, South Korea. E-mail addresses: [email protected] (H.S. Kim), [email protected] (B.M. Lee).

are commonly used in the cosmetic industry and personal care products as they rarely cause contact allergy in the skin (Lundov et al., 2009). The relative hypoallergenic and effective antibacterial properties of paraben lead to extensive uses in cosmetics: more than 35% of American cosmetic products, and more than 28% of Danish cosmetic products (Flyvholm, 2005; Steinberg, 2006). Parabens are found in many of the baby products across the entire marketplace and there has been growing concern over the possibility that they may be a risk to human health (Karpuzoglu et al., 2013). The major source of parabens exposure to humans was assumed to be topical application of personal care products and immediately excreted in the urine (Janjua et al., 2007). The daily exposure levels of total parabens is estimated to be 76 mg, with cosmetics and personal care products accounting for 50 mg (Cashman and Warshaw, 2005; Soni et al., 2005). Parabens have

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Please cite this article in press as: Kim, M.J., et al., Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.08.005

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M.J. Kim et al. / Regulatory Toxicology and Pharmacology xxx (2015) 1e8

been detected in urine, serum, breast milk, and human breast tumor tissue at a mean level of 20.6 ng/g tissue (Darbre et al., 2004; Frederiksen et al., 2011; Barr et al., 2012). However, some research results on parabens claimed that it can cause breast cancer or problems in male reproductive system similar to other endocrine disrupting chemicals (EDCs) (Harvey and Everett, 2006; Krogenæs et al., 2014; Yoon et al., 2014). Especially, as it is more harmful for infants and children, the Danish government banned its usage in products for children aged less than three and the EU recommended not to use it in products for infants aged less than six months (SCCS, 2013). The level of preservatives used in cosmetic and skincare products is usually less than 1%, and in most cases the typical usage is at levels 0.01e0.3%. Parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-, isopropyl-, or isobutylparaben) were reported to have endocrine disrupting effects in in vitro and in vivo experiments (Hu et al., 2013a). Parabens mimic estrogen by binding to estrogen receptors (ERs) on human breast cancer MCF-7 cells (Byford et al., 2002; Charles and Darbre, 2013; Routledge et al., 1998). In the uterotrophic assay, relative uterine weight was dose-dependently increased by administration of benzylparaben in female SpragueeDawley rats. The oral lowest observed effect dose (LOED) of benzylparaben was determined to be 0.16 mg/kg bw/day (Hu et al., 2013a). In addition, the LOEDs of methyl-, ethyl-, propyl-, and butylparaben were estimated to be 16.5, 6, 20, and 7 mg/kg bw/day respectively by oral administration in uterotrophic assay using immature CD-1 mice (Lemini et al., 2003). Isobutylparaben also exhibited an estrogen-like property, which may be mediated by a progesterone receptor (PR) and/or estrogen receptor a (ERa) signaling pathway (Vo and Jeung, 2009). In addition to estrogenic properties, parabens were reported to have androgenic activity, indicating that some parabens have adverse effects on the male reproductive system (Darbre and Harvey, 2008; Oishi, 2001, 2002a, 2002b, 2004). However, it is also a controversial point that paraben possesses low toxicity and does not affect humans adversely (Golden et al., 2005; Soni et al., 2005). No relation between exposure of paraben and estrogenic activity which affect the anogenital index of male offspring was found in an animal study by Shirai et al. (2013). Subcutaneous administration of parabens (2 mg/kg bw/ day) in neonatal rats produced no detectable testicular effects (Fisher et al., 1999). In addition, oral administration of isopropylparaben and isobutylparaben did not increase the incidence of cancer in animal experiments (Inai et al., 1985; Onodera et al., 1994). The safety of parabens in cosmetics and personal care products has been discussed at the political level in the EU in recent years. Danish Environmental Protection Agency (EPA) has prohibited the use of propylparaben and butylparaben in cosmetics for children under 3 years old since 2011 (DanishMOE, 2011). On the other hand, parabens are not included in the “negative list” in United States and in the “List of Prohibited and Restricted Cosmetic Ingredients” in Canada (Health Canada, 2014; U.S.FDA, 2000). The maximal concentration of 0.4% for single ester and 0.8% for mixture of esters is allowed in cosmetic products (MFDS, 2013; SCCS, 2010, 2013). Many toxicological studies have investigated four types of parabens (methyl-, ethyl-, propyl-, or butylparaben), but isobutylparaben (CAS No. 4247-02-3) and isopropylparaben (CAS No. 4191-73-5) have not been studied much. Furthermore, repeated dose dermal toxicity data was not available for risk assessment of isopropylparaben and isobutylparaben. In this study, a repeated dose dermal toxicity of isopropylparaben, isobutylparaben, or mixture of two substances was investigated in rats. The estimated systemic exposure dose of parabens through the use of cosmetic products, and risk assessment of isopropyl and isobutyl parabens in cosmetics were also studied.

2. Materials and methods 2.1. Chemicals and reagents Isopropylparaben (IPP, 99% pure, CAS No. 4191-73-5) and isobutylparaben (IBP, 99% pure, CAS No. 4247-02-3) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethanol (99% pure) was purchased from Merck KGaA (Darmstadt, Germany). 2.2. Study design The doses for animal treatment were selected based on assumptions regarding human exposure to parabens. The total daily use of cosmetics and toiletries containing paraben is assumed to be 10 g/day, based on the reports that about half of the average daily amount of product use (17.4e19.4 g/day) contains parabens as a preservative (CIR, 2008; KCII, 2012; SCCS, 2013). The maximum authorized concentration is 0.4%, which gives the maximal exposure of paraben at 4 mg/g. The calculation for the maximal daily exposure is: (daily exposure) ¼ (amount daily use)  (conc. in product) ÷ (weight of human), which gives approximately 0.7 mg/ kg. We used 60 kg for human weight. We then estimated the UF (uncertainty factor) at 300 [animal to human (10), human to human (10), an additional modifying factor for the use of subacute data (3)]. Therefore, the maximal daily level of paraben exposure comes to 210 mg/kg bw. The maximal daily dosage group in this experiment was determined to be 600 mg/kg bw, which is approximately three times higher than the estimated level of human exposure for a single paraben. One hundred and thirty rats were divided into 13 groups, with ten rats per group. Animal groups were applied dermally with IPP, IBP, or its mixture for 28 days. The first group was the control group; four groups were given IPP applied dermally at daily doses of 50, 100, 300, and 600 mg/kg bw, respectively; four groups were given IBP applied dermally at daily doses of 50, 100, 300, and 600 mg/kg bw, respectively; and four groups (Mix) were given a 1:1 mixture of IPP and IBP applied dermally at total daily doses of 100, 200, 600, 1200 mg/kg bw, as described in Appendix A. 2.3. Animals and treatment Male and female 5-week-old Sprague Dawley rats were purchased from Orient Bio (Sungnam, South Korea). The animals were housed in cages in a controlled environment with temperature at 25  C and under an illumination schedule of 12 h light/dark. As described previously (Martinez et al., 2014). The animals were fed 5L79 (Charles River rat and mouse 19% Auto) and water provided ad libitum. The protocol of the study followed current OECD Guidelines No. 410 on “repeated dose dermal toxicity: 21/28-day study” (OECD, 1981). Before the test, animals are randomized and assigned to the treatment and control groups. Shortly before testing, fur was clipped from the dorsal area of the trunk of the test animals. Shaving was carried out approximately 24 h before the test and repeat clipping or shaving was performed at approximately weekly intervals. Not less than 10 per cent of the body surface area was cleared for the application of the test substance. Groups of 10 male and 10 female rats were topically applied IPP, IBP, or mixture of IPP and IBP dissolved in 100 ml of ethanol (99%), 5 days per week for 28 days. The test substances were applied to the shaved dorsal skin of the animals. Each subject was administered the cream at 50 ml/cm2 of body surface area. Between applications the test substance is held in contact with the skin with a porous gauze dressing and nonirritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may

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be used to prevent ingestion of the test substance, but complete immobilisation is not a recommended method. The control group was given 99% ethanol. Animals were observed twice daily; clinical findings and body weights were recorded. Changes in body weight and the amounts of food or water consumption were recorded throughout the experiment (Data not shown). Prior to necropsy, animals were fasted overnight. Under anesthesia with diethyl ether, blood was collected via abdominal aorta. Liver, kidneys, heart, brains, testes, and prostates from male rats, and ovaries and vaginas from female animals were removed and weighed. The dorsal skin from the site of application was also removed from animals. The main lobe of liver, left kidney, sectioned brain, dorsal skin and heart were fixed with 10% neutral formalin solution. About 1 ml of blood sample was added in a heparin tube, and the remaining blood was centrifuged at 3000 rpm for 15 min to extract the serum. 2.4. Hematological evaluation Eight hematological parameters were analyzed in the blood sample: hemoglobin, hematocrit (Hct), red blood cell (RBC), white blood cell (WBC), platelet, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Concentrations of biochemical parameters measured in the serum were: albumin, glucose, total cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, calcium, phosphorus, triglyceride (TG), glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and gamma glutamyltranspeptidase.

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weight, food and water consumption were similar among all groups (Data not shown). The protocol for measuring body weights and feed/water consumption was based on the OECD guidelines for the Testing of Chemicals (OECD, 1981). Changes of body weights and feed/water consumption were measured twice a week. 3.2. Organ weights The relative weight of heart and kidneys increased in a dosedependent manner by simultaneous application of IPP and IBP in male rats. Significant difference (p < 0.05) in relative weight of kidney and testis was observed in male Mix 600 group, and heart in male Mix 300 and Mix 600 groups (Table 1). The higher the levels of parabens administered in male rats, the more weight gains of the left and right testes were observed. The relative weight of testes showed significant difference in Mix 600 group (p < 0.05). owever, significant difference in organ weights were not observed in female rats. The vagina and uterus, as a target organs for endocrine disruptors, did not show any significant change among groups (Appendix B). 3.3. Hematological analysis There was no significant difference in hematological evaluation. The concentrations of Naþ increased in a dose-dependent manner in male IBP (50 or higher) groups, and all male and female Mix groups. The concentration of Cl in the serum were significantly increased in female IBP 600, male IBP 300 and IBP 600, female Mix 300 and higher, and in all male Mix groups (Appendices CeF). 3.4. Evaluation of serum hormone concentrations

2.5. Histopathological evaluation Histological interpretation of the tissues was carried out at the Department of Veterinary Histology and Molecular Pathology, College of Veterinary Medicine, Kangwon National University (Chuncheon, Kangwon-do). Brains, hearts, kidneys, the biggest lobe of livers, and sectioned dorsal skin were fixed in a solution of 10% neutral formalin. The tissues were post-fixed and embedded in paraffin. Three to 5 mm thick sections were cut and stained with hematoxyline and eosin (H&E) for light microscopy. 2.6. Analysis of serum hormone concentrations Hormone levels were measured by enzyme-linked immunosorbent assay (ELISA). Triiodothyronine (T3), follicle-stimulating hormone (FSH), and estradiol concentrations were determined using rat ELISA kits manufactured from Cusabio® (Wuhan, China). Insulin level was measured using rat insulin ELISA kit manufactured from Shibayagi© (Shibukawa, Japan). The concentration of testosterone and thyroid-stimulating hormone (TSH) was assessed using rat/mouse ELISA kits of IBL-America©(Minneapolis, MN, USA). 2.7. Statistical analysis All data were statically analyzed with SPSS®12.0 KO for Windows (SPSS Inc.), using one-way ANOVA with Tukey's test followed by Duncan's test. 3. Results 3.1. Body weight, food and water consumption No animal died during the experimental period. Average body

There were no significant differences in serum T3, TSH, insulin, E2, or testosterone concentrations between control and isoparaben-treated groups of female rats (Data not shown). There were no statistical differences in FSH level in male rats between control and IPP or IBP-treated groups. In contrast, FSH concentration was dose-dependently decreased in groups treated with mixture of IPP and IBP, and statistically significant differences were observed from 100 Mix (a total of 200 mg/kg bw/day, IPP 100 mg/kg bw/day plus IBP 100 mg/kg bw/day) or higher groups (Table 2). Serum T3, E2, and testosterone levels were significantly decreased in rats treated with high dose of IPP and IBP mixture (Table 2). 3.5. Histopathological analysis Histopathological changes in brain, liver, kidney, heart and skin were evaluated in the animals of each group. No significant histopathological signs were observed in the brain, liver, heart, and kidney, although some lesions which can naturally occur were found. In the female rats, skin treated with the Mix 600 and higher showed inflammation, dermis, multifocal lesions; however skin treated with the lowest dose (Mix 50 mg/kg bw) of the mixture showed epidermal hyperplasia (Table 3). All IPP-treated groups did not show any specific lesions in either male or female rats (Fig. 1). Dose-dependent specific lesions were found in only female groups treated with IBP and the mixture of two isoparabens. Epidermal hyperplasia with hyperkeratosis was seen in one sample from IBP 100 group, and three each from the IBP 300 and IBP 600 groups. Pustule was found in one sample from IBP 100 and one from IBP 300, and three from IBP 600 groups. Minimal hyperkeratosis was found in one sample each from Mix 50 or 100; one minimal and one mild from Mix 300, and three samples from Mix 600 showed moderate epidermal hyperplasia with hyperkeratosis. Moderate pustule and

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Table 1 Mean relative organ weight of male rats treated in the skin with isopropyl paraben, isobutyl paraben, or mixture of two parabens. Groups

Brain (g)

Control IPP 50 IPP 100 IPP 300 IPP 600 IBP 50 IBP 100 IBP 300 IBP 600 Mix 50 Mix 100 Mix 300 Mix 600

2.025 2.076 2.046 2.066 2.086 2.039 2.039 2.042 2.062 2.033 2.066 2.094 2.160

± ± ± ± ± ± ± ± ± ± ± ± ±

Heart (g)

0.138 0.053 0.118 0.135 0.170 0.164 0.066 0.108 0.159 0.119 0.084 0.084 0.109

1.131 1.161 1.148 1.150 1.166 1.161 1.181 1.186 1.218 1.148 1.221 1.224 1.225

± ± ± ± ± ± ± ± ± ± ± ± ±

Liver (g)

0.075 0.049 0.051 0.078 0.059 0.073 0.045 0.074 0.072 0.098 0.054 0.036* 0.095*

12.446 12.788 12.746 12.630 12.523 12.708 12.628 12.603 12.131 12.463 12.427 11.522 11.506

± ± ± ± ± ± ± ± ± ± ± ± ±

Prostate (g) 1.095 0.954 0.806 1.562 0.678 0.473 0.725 1.524 0.774 0.968 0.482 1.039 0.861

0.453 0.462 0.443 0.453 0.460 0.433 0.417 0.444 0.442 0.466 0.458 0.463 0.507

± ± ± ± ± ± ± ± ± ± ± ± ±

Kidney (L)

0.071 0.067 0.041 0.088 0.090 0.072 0.065 0.106 0.088 0.079 0.148 0.055 0.125

1.157 1.178 1.168 1.172 1.156 1.178 1.176 1.207 1.212 1.165 1.192 1.190 1.285

± ± ± ± ± ± ± ± ± ± ± ± ±

Kidney (R)

0.046 0.036 0.056 0.052 0.045 0.071 0.083 0.070 0.048 0.056 0.052 0.072 0.101*

1.160 1.163 1.173 1.165 1.173 1.157 1.158 1.190 1.179 1.144 1.181 1.196 1.268

± ± ± ± ± ± ± ± ± ± ± ± ±

Testis (L)

0.056 0.053 0.036 0.069 0.103 0.075 0.048 0.061 0.046 0.041 0.044 0.035 0.105*

1.559 1.566 1.561 1.559 1.591 1.563 1.556 1.582 1.570 1.564 1.596 1.616 1.731

± ± ± ± ± ± ± ± ± ± ± ± ±

Testis (R)

0.123 0.166 0.124 0.175 0.150 0.143 0.096 0.127 0.088 0.176 0.093 0.099 0.103*

1.527 1.543 1.539 1.546 1.575 1.548 1.557 1.551 1.552 1.591 1.603 1.633 1.722

± ± ± ± ± ± ± ± ± ± ± ± ±

0.117 0.165 0.124 0.161 0.154 0.158 0.102 0.115 0.107 0.180 0.114 0.190 0.128*

Asterisk (*) indicates that significant difference compared with control (p < 0.05). Abbreviations: IPP (isopropyl paraben), IBP (Isobutyl paraben), Mix (mixture of isopropyl paraben and isobutyl paraben).

inflammation of dermis with multi focal was also seen in one sample of Mix 600 group (Fig. 2). In conclusion, the daily NOAEL (no observed adverse effect level) values for IPP and IBP for female skin damage were 600 and 50 mg/ kg bw/day, respectively. The LOAEL (lowest observed adverse effect level) value for mixture of two isoparabens was 50 mg/kg bw/day (a total of 100 mg/kg bw/day, IPP plus IBP) in female rats. 4. Discussion There have been growing concerns about parabens (including isopropyl paraben and isobutyl Paraben) due to their suspected endocrine disrupting effects. A series of studies on mice and rats have found that parabens may affect male reproductive system and estrogenic properties (Darbre and Harvey, 2008; Oishi, 2001, 2002a, 2002b, 2004). In vitro and in vivo studies showed that various kind of parabens (2-ethylhexyl-, heptyl-, benzyl-, butyl-, propyl-, ethyl-, and methylparaben) have estrogenic effects (Blair et al., 2000). And Watanabe et al. (2013) found that the size and bulkiness of the alkyl group of paraben tend to increase affinity for estrogen receptor (ER), which also implies that the use of parabens in combination may be more toxic than that of paraben individually. Vo and Jeung (2009) observed an increase in uterus weight and expression of uterine calbindin-D9k (CaBPe9K, a potent biomarker for screening estrogen-like environmental chemicals) in an immature rat model after parabens treatment. Moreover parabens induced an estrogen response element (ERE) related expression of CaBPe9K and PR mRNA in GH3 rat pituitary cells,

which resulted in significant increases in uterus weight in uterotrophic assays (Vo and Jeung, 2009; Vo et al., 2011, 2012; Yang et al., 2012). In addition, the adipogenic ability of parabens was reported in murine 3T3-L1 cells, and the potency increases as the length of alkyl chain in paraben increases (Hu et al., 2013b). However, there was no significant estrogenic effect of parabens shown in this experiment. Although antibacterial synergistic effects of several combinations of parabens have been well demonstrated, there have been only a limited number of toxicity studies regarding estrogenic or androgenic effect of multiple paraben exposure in vitro or in vivo. An acute oral toxicity study concluded that the toxicity of paraben mixtures did not show synergistic toxicity (Sado, 1973). A study indicated that exposure of paraben combination may induce synergistic effect on rat pituitary GH3 cells as the expression of CaBPe9K mRNA and protein, and PR-B gene increased more with multiple parabens (Yang et al., 2012). In the case of combinational exposure of parabens (methyl-plus ethylparaben, or methyl-plus propylparaben), the permeation rates through pig ear skin were significantly reduced (Caon et al., 2010). There is the possibility that a number of androgen receptor (AR) antagonists from various sources may act together at the receptor to produce joint effects even in very low concentrations (Orton et al., 2014). Therefore, the effect of combinational exposure of more than two parabens on human health needs to be further investigated. In order to obtain safe limits of paraben for humans, it is very important to know the absorption rate of paraben; it has been

Table 2 Serum hormone concentrations of male SpragueeDawley rats treated with isopropyl paraben, isobutyl paraben, or mixture of two parabens. Groups

T3 (ng/mL)

Control IPP 50 IPP 100 IPP 300 IPP 600 IBP 50 IBP 100 IBP 300 IBP600 Mix 50 Mix100 Mix300 Mix600

0.78 0.81 0.76 0.80 0.77 0.81 0.74 0.73 0.71 0.82 0.77 0.74 0.69

± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.06 0.04 0.06 0.05 0.07 0.03 0.06 0.09 0.04 0.10 0.09 0.07*

TSH (ng/mL) 2.09 2.19 2.01 1.81 1.61 1.95 2.16 1.73 1.64 1.95 1.75 1.83 1.53

± ± ± ± ± ± ± ± ± ± ± ± ±

0.55 0.70 0.48 0.38 0.52 0.41 0.57 0.66 0.32 0.39 0.68 0.21 0.36*

Insulin (ng/mL) 1.57 1.61 1.62 1.65 1.39 1.65 1.47 1.50 1.65 1.57 1.73 1.52 1.65

± ± ± ± ± ± ± ± ± ± ± ± ±

0.44 0.35 0.37 0.44 0.51 0.73 0.45 0.58 0.43 0.62 0.43 0.41 0.62

E2 (pg/mL) 15.41 14.75 16.03 15.60 13.49 14.39 13.86 15.12 13.78 13.27 12.59 13.22 12.95

± ± ± ± ± ± ± ± ± ± ± ± ±

3.29 2.34 3.16 2.95 2.69 1.39 2.12 2.59 2.85 1.34 3.36 1.78 1.98*

FSH (IU/L) 4.54 3.61 3.48 3.36 3.29 3.55 3.53 3.31 3.28 3.68 3.02 2.73 2.35

± ± ± ± ± ± ± ± ± ± ± ± ±

0.60 0.57 0.45 0.56 0.79 0.80 0.95 1.08 0.84 0.69 0.42* 0.23* 0.49*

Testosterone (ng/mL) 3.88 3.81 3.79 3.61 3.48 3.98 3.75 3.52 3.42 3.85 3.72 3.61 3.26

± ± ± ± ± ± ± ± ± ± ± ± ±

0.58 0.61 0.50 0.67 0.77 0.74 0.68 0.78 0.86 0.50 0.79 0.61 0.69*

Asterisk (*) indicates significant difference compared with the control (p < 0.05). Abbreviations: E2 ¼ estradiol; FSH ¼ follicle-stimulating hormone; IPP ¼ isopropyl paraben, IBP ¼ Isobutyl paraben; Mix ¼ mixture of isopropyl paraben and isobutyl paraben; T3 ¼ triiodothyronine; TSH ¼ thyroid-stimulating hormone.

Please cite this article in press as: Kim, M.J., et al., Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.08.005

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Table 3 Histopathology of skin of female SD rats treated with isopropyl paraben, isobutyl paraben or mixture of two parabens. Control group

Control

No specific lesion

3

Isopropyl paraben (IPP) group No specific lesion Inflammation, dermis, (multi)focal Minimal

IPP 50 3 0 0

IPP 100 3 0 0

IPP 300 3 0 0

IPP 600 3 0 0

Isobutyl paraben (IBP) group No specific lesion Epidermal hyperplasia with hyperkeratosis Minimal Mild Moderate Epidermal necrosis Mild Pustule Minimal Inflammation, dermis, (multi)focal Minimal

IBP 50 3 0 0 0 0 0 0 0 0 0 0

IBP 100 2 1 0 1 0 0 0 1 1 0 0

IBP 300 0 3 2 1 0 1 1 1 1 1 1

IBP 600 0 3 0 2 1 0 0 3 3 1 1

Mixture of IPP and IBP group No specific lesion Epidermal hyperplasia with hyperkeratosis Minimal Mild Moderate Epidermal necrosis Mild Moderate Pustule Minimal Mild Moderate Inflammation, dermis, (multi)focal Minimal Mild Moderate

Mix 50 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

Mix 100 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0

Mix 300 1 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0

Mix 600 0 3 0 0 3 1 0 1 1 0 0 1 1 0 0 1

Abbreviations: IPP ¼ isopropyl paraben, IBP ¼ isobutyl paraben, Mix ¼ mixture of isopropyl paraben and isobutyl paraben. *The number of samples used to perform the histopathological examination is 3 per group. Therefore, a number “3” means that all animals in group had histopathological lesions.

reported to be from 6 to 35% in an in vitro environment (Bando et al., 1997; Harville et al., 2007; Pedersen et al., 2007; Seko et al., 1999). It was found that methyl-, propyl-, and butylparaben are partially absorbed in SpragueeDawley rats (Aubert et al., 2012), and the rate of paraben absorption in human skin was found to be 3.7% (SCCS, 2010). Interestingly alcohol has been found to enhance dermal penetration of methyl paraben in Guinea pig skin in vitro (Darbre and Harvey, 2008; Kitagawa et al., 1997; Lakeram et al., 2007). Extent of paraben toxicity absorbed in the body via oral or dermal route may be related to how efficiently the tissue can metabolize paraben. Parabens have been shown to cause cytotoxicity and induce apoptotic action or have estrogenic activity, but paraben metabolites (hydrolyzed or conjugated) did not cause significant toxic effects in several in in vitro and in vivo studies (Dagher et al., 2012; Hossaini et al., 2000; Khanal et al., 2012;
us, 1998; Watanabe Martín et al., 2010; Nakagawa and Molde et al., 2013). Human carboxyesterase (hCE) expressed in human liver microsomes (hCE1), and colon and skin (hCE2) is a main paraben metabolic enzyme in human body (Dagher et al., 2012; Pindel et al., 1997; Zhu et al., 2005). UDP-glucuronyosyltransferase (UGT) is suspected to be an enzyme of paraben and glucuronidated form of paraben is found in human urine (Ye et al., 2006). Previous studies have reported that bioaccumulation or toxicity of paraben might be due to the difference of enzymes expressed in each cell. MCF-7 expresses hCE2 less than skin tissue does (Byun et al., 2008; Dagher et al., 2012). It is worthy to note that the increase of bulkiness of paraben decreases its absorption rate, which may be

another key to explain lower paraben toxicity than expected. Another interesting result of our data was the decrease in serum FSH level in rats treated with mixture of IPP and IBP. The decreased FSH level in the mixture of IBP and IPP group can be attributed to a direct effect of thyroid hormones. Previous study reported that thyroid gland weight was significantly increased in rats when exposed to MP and BP doses of 1000 mg/kg bw/day (Vo et al., 2010). Thyroid gland weight changes may be associated with activation of a negative feedback mechanism in response to decreased serum thyroid hormone levels (Zabka et al., 2011). In this study, any significant toxic effects were not observed in rats except macroscopic and microscopic histopathological changes in skin. There were no changes in body weight and food and water consumption, and no significant difference in relative organ weight. Analysis of hematological parameters and biochemical parameters showed that two isoparabens did not produce hematotoxicity, hepatotoxicity, or nephrotoxicity. The concentration of serum Cl and Naþions changed significantly, but the range was within the concentration of normal rats (Giknis and Clifford, 2006). Dermal application of isobutyl paraben over 100 mg/kg bw/day caused necrosis in epidermis, and consequent proliferating cells and pustules only in female rats. For a single paraben, isopropyl paraben was shown to be far less toxic than isobutyl paraben. The highest dose of 600 mg/kg bw IPP did not produce any toxic effects in rats, providing NOAEL value of 600 mg/kg bw in both sexes. However, the NOAEL of IBP was estimated to be 50 mg/kg bw in female rats. In this study, significant macroscopic damage to skin was seen in all animal groups (from 50 mg/kg bw Mix, 100 mg/kg bw as a total of IPP and

Please cite this article in press as: Kim, M.J., et al., Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.08.005

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Fig. 1. Histopathology of the skins of female rats treated with isobutyl paraben (IBP). A:IBP 50, B: IBP 100, C:IBP 300, D:IBP 600. In B, note the thickened epidermis with acanthosis (dark arrow). In C and D, note the hyperplastic epidermis (dark arrow) to which pustule (dark arrow) is attached. A, B, C, and D were graded as normal, mild, mild, and moderate for epidermal hyperplasia, respectively. H&E. Magnification ¼ x200.

IBP) treated with isoparaben mixture provided LOAEL of 100 mg/kg bw. It is also worth noting that isoparabens are being used less than other types of parabens (e.g., methyl paraben, ethyl paraben). Therefore, the dose selected for the mixture of isoparabens in

this study may be much higher than the levels of the mixture in practical use, which may lead to the overestimation of the mixture effects. Nonetheless, we can assume that isoparabens might be used at the maximum legal limits considering extreme cases.

Fig. 2. Histopathology of the skins of female rats treated with the mixture (Mix) of IPP and IBP. A: Mix 50, B: Mix 100, C: Mix 300, D: Mix 600. Figure A and B showed minimal epidermal hyperplasia in skin. C represents the thickened epidermis (dark arrow) with epidermal hyperplasia and hyperkeratosis. In D, note the severely injured skin with epidermal necrosis and inflammation (dark arrow). Also, note the epidermal epithelial cells (dark arrow) participating in the recovery process. H&E. Magnification ¼ x200.

Please cite this article in press as: Kim, M.J., et al., Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.08.005

M.J. Kim et al. / Regulatory Toxicology and Pharmacology xxx (2015) 1e8

5. Conclusion Although isoparaben mixture was shown to be more toxic than a single IBP treatment, the current maximum concentration of two isoparabens in the use of cosmetics was concluded to be safe. In fact, the dosage of “Mix” groups was based on current regulations stating that more than two parabens are allowed with concentrations of up to 0.8%, double that of a single paraben. Microscopic histopathological analysis clearly shows that the mixture of isoparabens impairs skin more synergistically in the animal study. Therefore, the synergic effect of two isoparabens whether lower dose or other combination of paraben mixture damages to skin more than a single paraben needs to be further investigated. In addition, why there is a gender difference with female rats being more susceptible to IBP should be elucidated. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by a grant (KFDA2012-17014) from Ministry of Food and Drug Safety (MFDS) in 2012. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.yrtph.2015.08.005. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2015.08.005. References Aubert, N., Ameller, T., Legrand, J., 2012. Systemic exposure to parabens: pharmacokinetics, tissue distribution, excretion balance and plasma metabolites of [14C]-methyl-, propyl- and butylparaben in rats after oral, topical or subcutaneous administration. Food Chem. Toxicol. 50, 445e454. Bando, H., Mohri, S., Yamashita, F., Takakura, Y., Hashida, M., 1997. Effects of skin metabolism on percutaneous penetration of lipophilic drugs. J. Pharm. Sci. 86, 759e761. Blair, R.M., Fang, H., Branham, W.S., Hass, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., Perkins, R., Sheehan, D.M., 2000. The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol. Sci. 54, 138e153. Barr, L., Metaxas, G., Harbach, C.A., Savoy, L.A., Darbre, P.D., 2012. Measurement of paraben concentrations in human breast tissue at serial locations across the breast from axilla to sternum. J. Appl. Toxicol. 32, 219e232. Byford, J.R., Shaw, L.E., Drew, M.G., Pope, G.S., Sauer, M.J., Darbre, P.D., 2002. Oestrogenic activity of parabens in MCF7 human breast cancer cells. J. Steroid Biochem. Mol. Biol. 80, 49e60. Byun, H.M., Choi, S.H., Laird, P.W., Trinh, B., Siddigui, M.A., Marquez, V.E., Yang, A.S., 2008. 2'-deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine: a novel inhibitor of DNA methyltransferase that requires activation by human carboxylesterase 1. Cancer Lett. 266, 238e248. ~es, C.M.O., 2010. EvalCaon, T., Costa, A.C.O., de Oliveira, M.A.L., Micke, G.A., Simo uation of the transdermal permeation of different paraben combinations through a pig ear skin model. Int. J. Pharm. 391, 1e6. Cashman, A.L., Warshaw, E.M., 2005. Parabens: a review of epidemiology, structure, allergenicity, and hormonal properties. Dermatitis 16, 57e66. Charles, A.K., Darbre, P.D., 2013. Combinations of parabens at concentrations measured in human breast tissue can increase proliferation of MCF-7 human breast cancer cells. J. Appl. Toxicol. 33, 390e398. CIR (Cosmetic Ingredient Review), 2008. Final Amended Report on the Safety Assessment of Methylparaben, Ethylparaben, Propylparaben, Isopropylparaben, Butylparaben, Isobutylparaben, and Benzylparaben as Used in Cosmetic Products. www.cir-safety.org/sites/default/files/PR427.pdf. Darbre, P.D., Aljarrah, A., Miller, W.R., Coldham, N.G., Sauer, M.J., Pope, G.S., 2004. Concentrations of parabens in human breast tumours. J. Appl. Toxicol. 24, 5e13. Darbre, P.D., Harvey, P.W., 2008. Paraben esters: review of recent studies of

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Please cite this article in press as: Kim, M.J., et al., Toxicological evaluation of isopropylparaben and isobutylparaben mixture in SpragueeDawley rats following 28 days of dermal exposure, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.08.005