Food and Chemical Toxicology 72 (2014) 40–50
`
`Contents lists available at ScienceDirect
`
`Food and Chemical Toxicology
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x
`
`Invited Review
`A review of the nonclinical safety of TranscutolÒ, a highly purified
`form of diethylene glycol monoethyl ether (DEGEE) used as a
`pharmaceutical excipient
`Dexter W. Sullivan Jr. a,⇑, Shayne C. Gad a, Marjorie Julien b
`
`a Gad Consulting Services, 102 Woodtrail Lane, Cary, NC 27518, USA
`b Gattefossé, 36 Chemin de Genas, 69804 Saint-Priest, France
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 8 April 2014
`Accepted 30 June 2014
`Available online 9 July 2014
`
`Keywords:
`Excipient
`Drug delivery
`Oral formulation
`Parenteral formulation
`Food additive
`Cosmetic ingredient
`
`Contents
`
`TranscutolÒ (Diethylene glycol monoethyl ether, DEGEE), CAS # 111-90-0, is commonly used as a vehicle
`in the formulation or manufacturing process of pharmaceuticals, cosmetics, and food additives. This
`paper presents unpublished nonclinical safety data using a form of DEGEE which includes a significantly
`decreased level of impurities, specifically ethylene glycol and diethylene glycol. It also reviews the history
`of use, regulatory status, and previously published toxicity data for DEGEE. The review supports that
`DEGEE is well tolerated across animal species and gender with toxicity occurring only at levels well above
`those intended for human use. At high levels of exposure, the kidney is identified as the critical target
`organ of DEGEE toxicity. DEGEE is negative for genotoxicity in in vitro and in vivo studies. Subchronic
`and chronic toxicity studies produced no reports of preneoplastic changes in organs, but the animal data
`is insufficient to allow a definitive opinion as to carcinogenicity. In silico data suggested that DEGEE is not
`carcinogenic or genotoxic. Developmental toxicity was seen in rats but only at levels 200 times greater
`than the estimated oral Permissible Daily Exposure Level of 10 mg/kg/day. The nonclinical data along
`with the long history of DEGEE use as a vehicle and solvent by multiple routes provide evidence of its
`safety. Furthermore, the novel data discussed herein provides evidence that toxicity previously associ-
`ated with high levels of DEGEE in nonclinical studies conducted prior to 1990 could possibly be attributed
`to the presence of significant amounts of ethylene glycol or other impurities.
`Ó 2014 Elsevier Ltd. All rights reserved.
`
`1.
`
`2.
`3.
`4.
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
`Identity and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
`1.1.
`Economic uses in marketed products with human exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
`Regulatory status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
`Safety evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
`4.1.
`Toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
`Local tissue tolerance (skin, eye, intravenous, and mucosal irritation, sensitization, hematocompatibility, and parenteral irritation). . . . 43
`4.2.
`4.3.
`Acute toxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
`4.4.
`Repeat-dose toxicity studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
`4.4.1.
`Previously published oral data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
`Previously published inhalation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
`4.4.2.
`4.4.3.
`Previously published intramuscular data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
`4.4.4.
`Previously unpublished studies conducted by Gattefossé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
`Reproductive and developmental toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
`External data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
`4.5.1.
`4.5.2.
`Previously unpublished studies conducted by Gattefossé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
`⇑ Corresponding author. Address: 102 Woodtrail Lane, Cary, NC 27518, USA.
`
`4.5.
`
`E-mail address: dwsullivanjr@gmail.com (D.W. Sullivan Jr.).
`
`http://dx.doi.org/10.1016/j.fct.2014.06.028
`0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
`
`AMN1053
`Amneal v. Almirall, LLC
`IPR2018-00608
`
`1
`
`

`

`D.W. Sullivan Jr. et al. / Food and Chemical Toxicology 72 (2014) 40–50
`
`41
`
`5.
`
`Genotoxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
`4.6.
`Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
`4.7.
`Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
`Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
`Transparency Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
`
`1. Introduction
`
`TranscutolÒ, purified diethylene glycol monoethyl ether
`(DEGEE, CAS No. 111-90-0),
`is an ethylene oxide derivative.
`Because of its characteristics as a strong solubilizer coupled with
`its low toxicity, DEGEE has a long history of safe use as a solvent
`in many products including pharmaceuticals, cosmetics, and food
`applications. Numerous independent nonclinical studies on the
`safety of DEGEE are available in the published literature. This paper
`seeks to evaluate the safety of DEGEE by reviewing the current
`published literature and adding previously unpublished data per-
`formed by Gattefossé to evaluate the safety of the purified com-
`pound, TranscutolÒ, as a pharmaceutical excipient. A brief review
`of the current uses and regulatory status of TranscutolÒ are also
`included.
`
`1.1. Identity and characterization
`
`Diethylene glycol monoethyl ether (DEGEE, CAS No. 111-90-0)
`is a clear, colorless, hygroscopic liquid with a mild pleasant odor
`(Fig. 1). It is produced by condensation of ethylene oxide and alco-
`hol, followed by a purification distillation (USP-NF, 2013). DEGEE is
`
`soluble in water and miscible in acetone, benzene, chloroform, eth-
`anol (95%), ether, and pyridine. It is partially soluble in vegetable
`oils and insoluble in mineral oils (Rowe et al., 2012). Table 1 sum-
`marizes the physiochemical properties of DEGEE.
`DEGEE has a wide variety of uses including pharmaceutical
`applications, as an indirect food additive for use in food, nutraceu-
`tical products and dietary supplements, and in cosmetics. The pri-
`mary supplier in the US for pharmaceutical grade DEGEE is
`Gattefossé using the trade name TranscutolÒ (Osborne, 2011). Prior
`to 1988, Gattefossé only produced a single grade of DEGEE mar-
`keted under the trade name TranscutolÒ that was 99.5% pure.
`However, current pharmaceutical grade TranscutolÒ P (topical
`route) and TranscutolÒ HP (oral route) are 99.8% and 99.9% pure,
`respectively. A cosmetics only grade TranscutolÒ CG is 99.5% pure.
`The identified impurities for pharmaceutical- and cosmetic-grade
`TranscutolÒ, along with their identified and maximum allowable
`(for pharmaceutical-grade) levels, are summarized in Table 2. It
`is important to note that industrial grades of the solvent are at best
`98% pure and contain significant levels of ethylene glycol and
`diethylene glycol as impurities. Toxicology studies prior to the
`1990s were typically performed with the industrial grade solvent,
`with many of the observed adverse effects being attributable to the
`ethylene glycol impurity (Osborne, 2011). More recent studies
`with the purified material provide further evidence suggesting
`the attribution of effects to the impurity.
`
`Fig. 1. Chemical structure of diethylene glycol monoethyl ether (C6H14O3, CAS No.
`111-90-0).
`
`2. Economic uses in marketed products with human exposure
`
`Table 1
`Physiochemical properties for DEGEEa.
`
`Property (Unit)
`
`Empirical formula
`Molecular weight
`Boiling point (°C)
`Freezing point (°C)
`Density (g/cm3)
`Vapor pressure (mmHg at 20 °C)
`Relative vapor density (air = 1)
`Flash point (°C)
`Octanol/water partition coefficient (log P)
`
`a Rowe et al. (2012).
`
`Value
`
`C6H14O3
`134.17
`198–201
`105 to 103
`0.988
`0.07–0.12
`4.6
`90–96.1
`0.54
`
`DEGEE has a long history of use in pharmaceutical applications
`worldwide in the United States of America, Asia, and Europe. It is
`an effective solubilizer and is used in oral, topical, transdermal
`and injectable human and veterinary pharmaceutical products. In
`recent years it has been widely used as a solvent for topical prod-
`ucts on account of three main properties: firstly, it has been shown
`to solubilize actives that are insoluble in common solvents such as
`propylene glycol and ethanol. Secondly, it modifies the skin pene-
`tration properties of active ingredients allowing different drug
`delivery outcomes to be obtained including enhanced local absorp-
`tion, a prolonged release depot effect or systemic absorption for
`transdermal applications (Osborne, 2011); lastly it provides func-
`tionality at concentrations which avoid safety and tolerability
`issues.
`
`Table 2
`Characterization of the impurities or accompanying contaminants for TranscutolÒ.
`
`Substance
`
`CAS No.
`
`ICH Q3C (ppm)
`
`USP36-NF31 (ppm)
`
`Max level in TranscutolÒ Gradesa (ppm)
`
`Ethylene Glycol
`Diethylene Glycol
`Ethylene oxide
`2-Methoxyethanol
`2-Ethoxyethanol
`
`107-21-1
`111-46-6
`75-21-8
`110-80-5
`109-86-4
`
`Class 2 (6620)
`–
`–
`Class 2 (650)
`Class 2 (6160)
`
`6620
`6150
`61
`650
`6160
`
`CG
`6620
`6250
`61
`650
`6160
`
`P
`6100
`6150
`61
`650
`6100
`
`HP
`620
`650
`61
`620
`650
`
`ppm: parts per million; CG is the cosmetics only grade TranscutolÒ and is 99.5% pure; P and HP are pharmaceutical grade TranscutolÒ and are 99.8% and 99.9% pure,
`respectively.
`a Source: Gattefossé SAS.
`
`2
`
`

`

`42
`
`D.W. Sullivan Jr. et al. / Food and Chemical Toxicology 72 (2014) 40–50
`
`Table 3
`DEGEE use as a vehicle in nonclinical studies.
`
`Species
`
`Route
`
`Duration
`
`Cat
`
`Intravenous
`
`1 month
`
`Rabbit
`
`Dermal
`
`Skin irritation
`28 days
`
`Ocular
`
`Rat
`
`Oral
`
`Mouse
`
`Oral
`
`Dog
`
`Oral
`
`Source: Gad et al. (2006).
`
`Eye irritation
`Eye irritation
`90 days
`Acute
`Fertility and embryo toxicity range-finding
`study
`Acute
`Chronic (12 months)
`90 days
`
`Dose
`
`DEGEE 2 mL/kg
`
`TranscutolÒ5 mL over 2 cm2 area
`TranscutolÒ 0, 300, 1000, 3000 mg/kg/
`day
`TranscutolÒ 0.1 mL
`TranscutolÒ 0.1 mL
`DEGEE 0%, 0.25%, 1% and 5%
`TranscutolÒ 5.0 g/kg
`DEGEE 500, 1000, 2000, 4000 mg/kg/
`day
`DEGEE
`DEGEE
`DEGEE
`
`Comments
`
`Well tolerated, no evidence of hemolysis or
`hematotoxicity
`50%; Non-irritant
`Undiluted; NOEL > 1000 mg/kg/day
`
`30%; Slight irritation
`Undiluted; Slight irritation
`NOEL is 1%
`LD 50 > 5000 mg/kg
`NOEL > 500 mg/kg/day
`
`6.6 g/kg tested toxic
`NOEL: 850–1000 mg/kg
`NOAEL: 1500 mg/kg/day
`
`In topical products DEGEE is often used in an aqueous gel. ACZ-
`ONE, the 5% dapsone gel for the treatment of acne was the first pre-
`scription drug product containing DEGEE as TranscutolÒ approved
`by the FDA (Osborne, 2011). TranscutolÒ has also been formulated
`in solutions, ointments and creams (emulsions and microemul-
`sions) for the delivery of hormones, anti-inflammatory, anti-fungal,
`anesthetic, analgesic and antiseptic agents in prescription products
`approved in numerous countries around the world (USFDA CDER,
`2013; Gattefossé SAS).
`In Europe, TranscutolÒ HP (the high purity DEGEE) has been
`used in a number of oral prescription drugs including the oral drop
`product ‘Lysanxia’, and the oral solutions ‘Pilosuryl’ and ‘Urosi-
`phon’ as well as a sublingual solution ‘Natispray’. In emerging Asia
`Pacific countries, notably South Korea, TranscutolÒ is used in soft
`gelatin capsules in approved antiviral, anti-inflammatory, and
`immune suppressant medicines (source Gattefossé SAS).
`Historically, TranscutolÒ has been used in injectable products,
`although its use in marketed human medicines remains limited
`to a few examples. In 1977 it was used in an intravenous injectable
`(IV) product ‘Trombovar’ approved in Europe for the treatment of
`varicose and spider veins in the leg; this product is no longer avail-
`able. More recently, it has been formulated in an IV and IM injec-
`tion of
`sodium diclofenac
`and an alpha beta-arteether
`intramuscular (IM) injection for the treatment of severe/cerebral
`malaria approved in India (source Gattefossé SAS).
`The aforementioned uses of high purity DEGEE in the form of
`TranscutolÒ are associated with human medicine. Veterinary med-
`icines require the same level of purity of excipients as human med-
`icines, and as such, TranscutolÒ is also widely used in veterinary
`applications including topical solutions, sprays and spot-on’s, often
`containing anti-parasitic agents which are formulated for transder-
`mal delivery (source Gattefossé SAS). It is also used in injectable
`veterinary products including the anti-inflammatory SC and IM
`product ‘Tolfedine’ and a ‘Vitamin E’ IV injection (Strickley, 2004).
`DEGEE is used as an indirect food additive for use in food, nutra-
`ceutical products and dietary supplements. The safety of use of this
`substance in such applications has been evaluated and is largely
`confirmed by many years of use.
`DEGEE has a long history of use in cosmetic and personal care
`applications. Currently, it can be found in over 740 cosmetic prod-
`ucts including eye makeup, fragrances, nail preparations, sunless
`tanning products, hair coloring products, and skin care prepara-
`tions (Elder, 1985; Osborne, 2011). The safety of use of this sub-
`stance in such applications has been evaluated and is largely
`confirmed by many years of use (CIR Expert Panel, 2006; Elder,
`1985; Osborne, 2011).
`DEGEE is used as a vehicle for use in in vivo nonclinical safety
`assessment studies and in clinical products. Gad et al. (2006)
`
`conducted a data mining project to determine the safe dosing level
`of drug delivery vehicles for in vivo animal studies. The results
`included information on 65 different vehicles and 9 animal species.
`The use of TranscutolÒ as a vehicle was reported for five species of
`animals and across four routes of exposure as shown in Table 3.
`
`3. Regulatory status
`
`The United States Food and Drug Administration (Center for
`Drug Evaluation and Research (CDER)) maintains an Inactive Ingre-
`dients Database (USFDA CDER, 2013). This database provides a par-
`tial
`listing of excipients being used in authorized medicinal
`products in the USA. This information can be used by industry as
`an aid in developing drug products. Once an inactive ingredient
`has appeared in an approved drug product for a particular route
`of administration, the inactive ingredient is considered qualified
`at the approved level and may require a less extensive review
`the next time it is included in a new drug product. For example,
`after an inactive ingredient has been approved for a specific dosage
`form and potency, a sponsor could consider it safe for use in a sim-
`ilar manner for a similar type of product. DEGEE is listed in the FDA
`Inactive Ingredient Database for topical use in a gel (25% maximum
`potency), transdermal use in a gel (5% maximum potency) and for
`use in a transdermal patch (maximum potency not reported). It is
`important to note that the approved maximum potency is not a
`limit for inactive ingredients, as higher levels may be approved
`with justification, but merely lists the amount of such ingredients
`that are currently approved for use in drug products.
`Similarly, DEGEE is listed in the Australian Register of Therapeu-
`tic Goods (Australian Therapeutic Goods Administration, 2013)
`which includes all therapeutic goods, including medicines and
`medical devices, approved for use in Australia. Health Canada
`(2013) maintains a repository of approved medicinal and non-
`medicinal ingredients approved for use in Canada. DEGEE is listed
`in the Canadian natural health products ingredients database.
`US FDA has approved DEGEE as an inactive ingredient for use as
`a component of adhesives for use in packaging, transporting, or
`holding food (21 CFR 175.105). US FDA has also approved DEGEE
`for use as a component of paper and paperboard in contact with
`dry food (21 CFR 176.180) and as a sanitizing agent for food-pro-
`cessing equipment and utensils (21 CFR 178.1010). The Joint
`FAO/WHO Expert Committee on Food Additives (JECFA) have eval-
`uated the use of DEGEE in food. The JECFA concluded that an
`Acceptable Daily Intake (ADI) for DEGEE could not be established
`due to the absence of adequate long-term (chronic/carcinogenic-
`ity) feeding studies in rats and mice and the absence of adequate
`data indicating that human intake of DEGEE from food are suffi-
`ciently low (JECFA, 1995).
`
`3
`
`

`

`D.W. Sullivan Jr. et al. / Food and Chemical Toxicology 72 (2014) 40–50
`
`43
`
`DEGEE has been evaluated by the Cosmetic Ingredient Review
`(CIR) expert panel (CIR Expert Panel, 2006; Elder, 1985). The panel
`noted that DEGEE was used in 80 different cosmetic preparations
`in 1981 (0.1 to greater than 50%) with the largest uses found in hair
`dyes and colors as well as skin cleansing creams, lotions, liquids,
`and pads. By 2002, DEGEE was used in 622 preparations at concen-
`trations ranging from 0.0004% to 80%. The panel concluded that
`based on the available data DEGEE is safe as presently used in cos-
`metics (2006; Elder, 1985). The Scientific Committee on Consumer
`Safety (SCCS, 2013) issued an opinion on the safety of DEGEE in
`cosmetic products. The SCCS concluded that DEGEE in cosmetic
`products (excluding oral hygiene and eye products) does not pose
`a risk to consumer health at concentrations up to 10% in rinse-off
`products, up to 7.0% in hair dye formulation and up to 2.6%, pro
`in all other cosmetic products provided that the level of ethylene
`glycol in DEGEE used is <0.1%.
`
`4. Safety evaluation
`
`A number of toxicity studies have been conducted with DEGEE
`by multiple routes of administration in a variety of species for a
`period of up to two years. Additionally, Gattefossé has completed
`a full battery of additional studies on DEGEE (as TranscutolÒ),
`including those evaluating toxicokinetics, local tolerance, skin sen-
`sitization, reproductive effects, teratogenicity, genotoxicity, and
`systemic toxicity. The new data for DEGEE generated within Gat-
`tefossé, in combination with the preexisting data, are presented
`here and serve to provide a dataset sufficient for determining the
`safety of DEGEE in humans.
`
`4.1. Toxicokinetics
`
`The absorption of DEGEE in vitro has been evaluated in
`human abdominal, whole skin. The rate of absorption was
`0.125 ± 0.103 mg/cm2/h,
`the
`permeability
`constant was
`1.32  104 cm/h, and the damage ratio (a measure of integrity
`of the skin) was 1.20 ± 2.62. Among numerous glycol ethers tested
`in this study, including ethylene glycol monomethyl, monoethyl
`and monobutyl ethers, and DEGEE and diethylene glycol butyl
`ether, DEGEE had the lowest damage ratio and the second lowest
`permeability constant and rate of absorption, suggesting a
`decreased absorption rate with increasing molecular weights of
`glycol ethers (Dugard et al., 1984).
`Unlike monoethylene glycol ethers, diethylene glycol ethers
`(including DEGEE) are poor substrates for alcohol dehydrogenase
`and expected to be good substrates for cytochrome P-450 based
`on experiments that measured induction of P-450. In an in vitro
`system using equine liver alcohol dehydrogenase, the Vmax (lmol)
`Km (lM) and Vmax/Km were 6.94, 6.31  102, and 0.11, respec-
`tively (Miller, 1987). In an adult human volunteer (sex and age
`not reported) given a single oral dose of 11.2 mmol DEGEE, approx-
`imately 68% of the dose was recovered in the urine as (2-ethoxy-
`ethoxy) acetic acid within 12 h (Kamerling et al., 1977).
`A number of studies have been completed in which multiple
`parameters were measured to further evaluate the toxicokinetic
`profile of purified DEGEE as TranscutolÒ. In an in vitro study per-
`formed to determine the metabolism profile of DEGEE (as Transcu-
`tolÒ) and ethylene glycol monoethyl ether (EGEE) formed by rat
`and human hepatocytes, EGEE was readily metabolized by both
`rat and human hepatocytes to ethoxy acetic acid (EAA) and ethyl-
`ene glycol (EG), and the rat liver cells metabolized EGEE at a higher
`rate than human liver cells. However, contrasting results were seen
`with DEGEE including slow metabolism by rat hepatocytes to sev-
`eral different unidentified metabolite peaks that accounted for
`
`approximately 1–17% of the total radioactivity. DEGEE was not sig-
`nificantly metabolized by human hepatocytes (Gattefossé, 2001a).
`In vivo, the absorption, distribution and excretion of DEGEE (as
`TranscutolÒ) was investigated comparably in Sprague–Dawley and
`BDIX rats after a single oral or intravenous dose of 20 mg 14C-
`DEGEE/kg bw each. The GLP-compliant study was performed
`according to internal laboratory methodology comparable to OECD
`417. Rapid excretion of radioactivity occurred in the urine, regard-
`less of sex and route of administration. The maximum plasma con-
`centration of the radioactivity was observed 0.25 h following
`intravenous injection, while after oral administration it was
`observed at 0.25–0.50 h post dose. The plasma half-life corre-
`sponded to 37–84 h with measurable concentrations observed in
`most of the tissues 168 h following administration. The absolute
`bioavailability of the radioactivity was very high (79–95%). The dis-
`tribution of radioactivity in tissues was characterized by high con-
`centrations detected in pituitary, thyroid, adrenals and bone
`marrow with regards to the concentrations observed in blood/
`plasma (100–1000 times less) at the same sampling time. The
`radioactivity levels in tissues was significantly decreased at 48 h.
`No biologically relevant differences were observed between the
`two rat strains (Gattefossé, 2002a).
`In studies evaluating the metabolic fate and excretion of DEGEE
`(as TranscutolÒ) results indicated that following a single oral
`administration, the large majority (90%) of the administrated
`radioactivity was rapidly excreted (within the first 24 h) in the
`urine and 14C-DEGEE was intensively metabolized as Ethoxyeth-
`oxyacetic acid (83%) and Diethylene glycol (5.4%) with only 3% of
`the urinary excreted radioactivity corresponding to unchanged
`compound.
`In plasma, only Ethoxyethoxyacetic
`acid and
`unchanged 14C-DEGEE were detected, which was consistent with
`urinary results. The GLP-compliant study was performed according
`to internal
`laboratory methodology comparable to OECD 417
`(Gattefossé, 2003).
`
`4.2. Local tissue tolerance (skin, eye, intravenous, and mucosal
`irritation, sensitization, hematocompatibility, and parenteral
`irritation)
`
`The current published data has shown that DEGEE is not a skin
`irritant in rabbits even after prolonged and repeated contact under
`normal study conditions while being only slightly irritating to rab-
`bit skin with the use of an occlusive wrap (Cragg, 2012; Rowe,
`1947; Krasavage and Terhaar, 1981). In rabbits, ocular administra-
`tion of 500 mg DEGEE has produced moderate irritation
`
`Table 4
`DEGEE acute toxicity summary (external publications).
`
`Route
`
`Oral
`
`Intravenous
`
`Intraperitoneal
`
`Subcutaneous
`
`Species
`
`Rabbit
`Mouse
`Rat
`Guinea Pig
`
`Cat
`Dog
`Rat
`Mouse
`Rabbit
`
`Rat
`Mouse
`
`Rat
`Mouse
`Rabbit
`
`Effect
`
`LD50 = 3620 mg/kg
`LD50 = 7250 mg/kg
`LD50 = 7500 mg/kg
`LD50 = 3000 mg/kg
`LDLo = 1000 mg/kg
`LD50 = 3000 mg/kg
`LD50 = 4000 mg/kg
`LD50 = 4300 mg/kg
`LD50 = 2500 mg/kg
`LD50 = 6300 mg/kg
`LD50 = 3900 mg/kg
`LD50 = 2300 mg/kg
`LD50 = 6000 mg/kg
`LD50 = 5500 mg/kg
`LD50 = 2000 mg/kg
`
`Source: Leadscope Portal Dataset, Version 3.1.2-1, Accessed March 18, 2014.
`
`4
`
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`

`44
`
`D.W. Sullivan Jr. et al. / Food and Chemical Toxicology 72 (2014) 40–50
`
`Table 5
`Acute toxicity studies conducted with TranscutolÒ (previously unpublished data from studies conducted by Gattefossé).
`
`Study type/duration
`
`Route
`
`Acute toxicity
`
`Oral (gavage)
`
`Species
`
`Rat
`
`Acute toxicity (Dose escalating)
`
`Oral (gavage)
`
`Dog
`
`Acute toxicity (Dose escalating)
`
`IV bolus (tail vein) Mouse
`
`Test article
`TranscutolÒ
`Pure (undiluled)
`Dose levels: 5000 mg/kg
`TranscutolÒ
`Pure (undiluled)
`Dose levels: 500, 1000, 1500, 2000 mg/kg
`TranscutolÒ
`Vehicle: Physiological saline solution
`M: 25, 50, 100, 200, 400, 800, 1600, 6400, 3200 and 4800 mg/kg
`F: 25, 50, 100, 200, 400, 800, 1600, 8000, 6400, 4800 and 3200 mg/kg
`
`Results/conclusion
`
`LD50(oral) > 5000 mg/kg
`
`MTD(oral) > 2000 mg/kg
`
`MTD(IV): 3200 mg/kg
`
`(Cragg, 2012; Union Carbide Corporation, 1968). When used as in
`vaginal and nasal gels and emulsions in rabbits with repeat doses,
`it has not shown itself to be an irritant (Mourtas et al., 2010;
`Elshafeey et al., 2009).
`GLP-compliant primary irritation single patch and a repeat
`insult patch tests in human performed by Gattefossé (1992,
`1993) showed that undiluted DEGEE (as TranscutolÒ) applied
`under occlusive conditions was well tolerated and did not lead to
`any classifiable primary or cumulative skin irritation. A skin irrita-
`tion study in rabbits, using an older, less pure form of DEGEE (as
`TranscutolÒ), which was not performed under GLP conditions but
`exceeded the current guideline requirements (OECD 404) in
`respect to animal numbers and can be considered as scientifically
`valid, showed that a 50% aqueous solution was not a skin irritant
`(Gattefossé, 1974). Guideline-conforming (OECD 405, EEC 92/69)
`eye irritation studies in rabbits performed under GLP conditions,
`revealed only a slight irritant effect to the eyes, when tested neat
`or as 30% aqueous solution (Gattefossé, 1996a). However, as the
`observed findings were only slight and transient in nature, and
`were not sufficient to be considered an eye irritant according to
`EU classification criteria (mean score of 2.00 for acute ocular irrita-
`tion), it is concluded that DEGEE (as TranscutolÒ) is not an eye
`irritant.
`Intravenous administration of 1 mL/kg or less of aqueous solu-
`tions containing concentrations of 5% or less is not hemolytic. In
`vitro hemolysis studies of a range of excipients showed no hemo-
`lysis caused by DEGEE (as TranscutolÒ) at concentrations up to
`80 ll/ml (Aparicio et al., 2005.) Intramuscular injection of 30% oily
`solution and 50% aqueous solutions of DEGEE (as TranscutolÒ)
`causes moderate but reversible irritation. Microemulsions contain-
`ing DEGEE (as TranscutolÒ) have been shown to not be irritating to
`veins when given intravenously (He et al., 2010).
`
`4.3. Acute toxicity studies
`
`Both external publications (see Table 4) and studies conducted
`by Gattefossé (see Table 5) indicate that the acute toxicity of
`DEGEE after oral, intraperitoneal, intravenous, and subcutaneous
`application can be regarded as very low in all species investigated.
`The LD50 values for acute toxicity were generally much higher than
`2000 mg/kg bw, and the available LC50 value for acute inhalation
`was >5 mg/L (i.e. 5.24 mg/L).
`
`4.4. Repeat-dose toxicity studies
`
`4.4.1. Previously published oral data
`A six week study was conducted in which groups of 10 male
`Sprague–Dawley rats were administered DEGEE by oral gavage at
`doses of 1340, 2680, and 5360 mg/kg/day. In the high dose group,
`four animals died before study termination and 3 were terminated
`moribund. Seven animals had bloody urine at various times
`
`throughout the study. Several other hematological and clinical
`chemistry signs were observed. One death also occurred at the
`intermediate dose prior to study termination. Lethargy was noted
`during the first week of treatment. However, there were no signif-
`icant effects of treatment with the intermediate dose on hematol-
`ogy or clinical chemistries. Increased organ weights seen include
`the relative liver, heart, and kidney weights (but not absolute
`weights of these organs) with respect to control. Microscopic
`changes included hyperkeratosis of the stomach (2/10), and splenic
`congestion (1/9). Because no effects were seen at the lowest dose,
`the NOAEL was established as 1340 mg/kg/day (European
`Chemicals Bureau, 2000; OECD, 2005).
`In a further study, groups of 15 male and female CFE rats were
`fed DEGEE at doses 250 and 2500 mg/kg bw (0.5% and 5.0% in the
`diet, respectively) for 90 days. Effects observed at the high dose
`included reductions in growth rate and food consumption as well
`as the average male final body weight. Decreased hemoglobin con-
`centration of high dose males was seen at 90 days and the hemo-
`globin concentration and red blood cell count were decreased in
`females at 45 days. In high dose males and females, oxalate crystals
`in urine were observed. Increased relative kidney weights were
`seen in high dose males and females and the spleen and thyroid
`of high dose females were increased. Advanced intracellular edema
`(hydropic degeneration) of the kidney was reported in 6 high dose
`males and 1 high dose female. Calcification of the renal cortex was
`reported in three high dose males and 1 high dose female. Based on
`these effects, the NOAEL was determined to be 250 mg/kg bw
`(Gaunt et al., 1968).
`Groups of 12 male and 12 female Wistar rats received diet con-
`taining 0%, 0.25%, 1.0%, and 5.0% DEGEE for 13 weeks. Decreased
`growth of male and female rats, which was associated with a
`reduction in food consumption, was seen in high-dose rats. No
`hematological changes were seen in any dose group. Males and
`females given 5% test material had elevated urinary glutamic-oxa-
`loacetic transaminase and kidney weights compared to controls.
`High dose males also had proteinuria. Hydropic degeneration
`was seen in the kidneys of two high dose males and one high dose
`female. Slight to moderate fatty changes in the liver were seen in
`most high dose animals (incidences not provided). Because no
`treatment-related effects were seen in 0.25% or 1.0% dose groups,
`the NOAEL in this study was 1.0% in the diet corresponding to
`about 800 mg/kg bw (Hall et al., 1966).
`Wistar rats were exposed orally to a blend of Labrasol, Labrafil,
`and TranscutolÒ (L/L/T) at dose levels of 0, 5, 10, or 20 mL/kg/day
`(approximately 0, 1000, 2000, and 4000 mg/kg/day TranscutolÒ)
`for four weeks to evaluate the safety of the formulation for use
`in in vivo non-clinical safety assessment studies for poor water sol-
`uble drugs. The blend was well tolerated at 5 mL/kg/day. In the
`mid-dose group, changes in appearance and behavior were seen.
`Lethality occurred in one animal at 20 mL/kg/day. In addition, renal
`and hepatic effects were also seen at 20 mL/kg/day. The authors
`
`5
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`D.W. Sulliva