Abstract
The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 26 apple-derived ingredients, which reportedly function mostly as skin conditioning agents in cosmetic products. Because apple-derived ingredients may be obtained from different apple cultivars, the composition of ingredients derived from different cultivars should be similar to that of ingredients reviewed in this safety assessment. Additionally, industry should continue to use good manufacturing practices to limit impurities that could be present in botanical ingredients. The Panel reviewed the available data to determine the safety of these ingredients and concluded that 21 of these ingredients are safe in cosmetics in the present practices of use and concentrations described in this safety assessment. However, the Panel also determined that the available data are insufficient to determine the safety of Pyrus Malus (Apple) Root Extract, Pyrus Malus (or Malus Domestica) (Apple) Stem Extract, Malus Domestica (Apple) Callus Extract, and Malus Domestica (Apple) Oil.
Keywords
Introduction
The safety of the following 18 • Pyrus Malus (Apple) Fruit Extract • Pyrus Malus (Apple) Bark Extract • Pyrus Malus (Apple) Carpel Powder • Pyrus Malus (Apple) Fiber • Pyrus Malus (Apple) Flower Extract • Pyrus Malus (Apple) Fruit • Pyrus Malus (Apple) Fruit Water • Pyrus Malus (Apple) Juice • Pyrus Malus (Apple) Leaf Extract • Pyrus Malus (Apple) Pectin Extract • Pyrus Malus (Apple) Peel Extract • Pyrus Malus (Apple) Peel Powder • Pyrus Malus (Apple) Peel Wax • Pyrus Malus (Apple) Pulp Extract • Pyrus Malus (Apple) Root Bark Powder • Pyrus Malus (Apple) Root Extract • Pyrus Malus (Apple) Seed Extract • Pyrus Malus (Apple) Stem Extract • Malus Domestica (Apple) Fruit Extract • Malus Domestica (Apple) Fruit Water • Malus Domestica (Apple) Fiber • Malus Domestica (Apple) Juice • Malus Domestica (Apple) Oil • Malus Domestica (Apple) Stem Extract • Malus Domestica (Apple) Fruit Cell Culture Extract • Malus Domestica (Apple) Callus Extract
The definitions of these ingredients are included in the
The following functions of
As stated above
Some of the ingredients (e.g., fruit/fruit-derived) reviewed in this safety assessment may be consumed as food, and daily exposure from food use would result in much larger systemic exposures than those from use in cosmetic products. The primary focus of the safety assessment of these ingredients as used in cosmetics is on the potential for effects from topical exposure.
The Panel has evaluated the safety of
In the current safety assessment, if a substance tested in a study is not clearly a cosmetic ingredient, because of the absence of information on the genus and species from which the substance was derived and/or the method of extraction used, the test substance will be referred to by pyrus malus or a common name (e.g., apple, apple juice, apple fruit extract, apple seed extract).
Data on procyanidin B-2 (epicatechin-(4β→8)-epicatechin, found in apple fruit) 9 are also included for use in this safety assessment. Procyanidins are members of the procyanidin or condensed tannins class of flavonoids. 10 Procyanidin B-2 is not a cosmetic ingredient.
Apple-derived ingredients may be obtained from different cultivars. Accordingly, the Panel noted that the composition of ingredients derived from different cultivars should be similar to the composition of ingredients reviewed in this safety assessment.
Data relating to effects on melanogenesis are included in this safety assessment. However, a review of ingredients for drug effects (i.e., noncosmetic effects) is not within the Panel’s purview.
Chemistry
Definition and Structure
Pyrus Malus
Definitions and Reported Functions of the Ingredients in this Safety Assessment. 1
aSkin bleaching agent is not regarded as a cosmetic use in the US,3 and the Panel did not evaluate safety for that use.
Chemical and Physical Properties
Pyrus Malus (Apple) Fruit Extract
Method of Manufacture
Pyrus Malus (Apple) Fiber
Information provided by the cosmetics industry indicates that Pyrus Malus (Apple) Fiber is derived from residue formed during apple juice production. The residue is dried and mechanically ground and sieved. 11
Pyrus Malus (Apple) Fruit Extract
Pyrus Malus (Apple) Fruit Extract has been prepared according to the following procedure: 12 Pyrus malus fruit was peeled manually and the pericarp was separated, dried at room temperature (26 - 28°C), and powdered. The powder was extracted with 95% ethanol. The ethanol was evaporated using a rotary evaporator, and the fruit extract was stored at −20°C prior to use.
According to information provided by the cosmetics industry, Pyrus Malus (Apple) Fruit Extract is made by extracting apples with a 50% aqueous 1,3-butylene glycol solution. 13 The extract is then treated as follows: filtration, sedimentation, filtration, adjustment of the concentration with additional 50% aqueous butylene glycol, and packaging.
The method of manufacture of a trade name material containing 10 - 25% Pyrus Malus (Apple) Fruit Extract (provided by the cosmetics industry) has been described as follows: 6 The plant material is extracted with 1,2-propylene glycol at “considerate” temperatures during a fixed time, and is sterile-filtered at the end of the fabrication. Lactic acid (.1 - 1%) is used for pH regulation, and the product contains .6% Bactiphen 250G (phenoxyethanol (75 - 100%), methylparaben (10 - 25%), ethylparaben (1 - 5%), propylparaben (1 - 5%), and butylparaben (1 - 5%)) as the preservative. Information provided by the cosmetics industry indicates that another trade name material containing 10 - 25% Pyrus Malus (Apple) Fruit Extract is manufactured using the same procedure, except that glycerin (vegetable origin) is the extraction solvent. Again, lactic acid (.1 - 1%) is used for pH adjustment, but the product contains potassium sorbate (.35%) and sodium benzoate (.35%) as preservatives.
According to information provided by the cosmetics industry, the process of manufacturing a trade name material containing 20% Pyrus Malus (Apple) Fruit Extract begins with the mechanical grinding/milling of pyrus malus fruit. 14 This is followed by extraction in butylene glycol (at specific pH and temperature), the addition of phenoxyethanol, and filtration. Another tradename material containing 20% Pyrus Malus (Apple) Fruit Extract is manufactured differently. 15 Processing (mechanical grinding and milling), is followed by extraction in propylene glycol (at specific pH and temperature) and, then, filtration.
The method of manufacture of a product described as an aqueous solution containing 20% Pyrus Malus (Apple) Fruit Extract is: 16 (1) solubilization of apple powder in water; (2) separation of soluble and insoluble phases by filtration; and (3) sterilizing filtration.
A product containing Pyrus Malus (Apple) Fruit Extract (18.5%), water (81.445%), and potassium sorbate (.055%) is manufactured as follows:
16
(1) solubilization of fruit of
The method of manufacture of a Pyrus Malus (Apple) Fruit Extract trade name material (identified as apple polyphenol extract) has been described as follows: 17 Unripe apples were crushed and pressed while 10% sodium metabisulfite solution was added. Pectolytic enzyme was used to clarify the juice obtained, and the mixture was centrifuged and/or filtered with diatomaceous earth. The clarified juice was passed through a column with aromatic synthetic adsorbents, after which the column was washed with distilled water to remove sugars and organic acids. Apple polyphenol extract was eluted with approximately 50% ethanol and concentrated using an evaporator. The concentrated fraction was dried (using a spray drier) to obtain the apple polyphenol extract as a brown powder. Toxicity data on this material (apple polyphenol extract (brown powder)) are summarized in the Toxicology section of this safety assessment.
Malus Domestica (Apple) Fruit Water
Malus Domestica (Apple) Fruit Water is prepared by dehydrating the fruit under vacuum at low temperature. 18
Apple Juice (genus and species of apple not stated)
An apple juice (from Fuji and Gala varieties; genus and species not stated) was obtained by mechanical pressure, depectinized in a water bath (2 h at 45°C), filtered through paper, bottled, closed, and stabilized by freezing. 19
Pyrus Malus (Apple) Pectin Extract
A simple procedure for the extraction of Pyrus Malus (Apple) Pectin Extract has been described as follows: 20 Approximately 40 g of fruit were washed with normal saline (.90% w/v sodioum chloride in water)], after which the fruit sample was crushed and homogenized in a blender. The resulting materials were allowed to dry in a hot air (60°C) oven for approximately 2 h until the pectin extracted turned into powder. To obtain sterilized pectin, the powdered pectin was sterilized with normal saline.
Apple Peel Extract
Prior to analysis for anthocyanin content, apples (
Pyrus Malus (Apple) Root Bark Powder
According to information provided by the cosmetics industry, Pyrus Malus (Apple) Root Bark Powder is extracted with carbon dioxide, and is then purified.
22
Another source indicates that the air-dried, powdered bark of
Pyrus Malus (Apple) Seed Extract
Pyrus malus seed extract has been prepared according to the following procedure: 24 After 3 months of stratification (steady exposure to moist environment before germination becomes possible), the seeds (not dehydrated) were extracted. The extraction procedure was performed using chilled 80% redistilled methanol, to which butylated hydroxytoluene (BHT, 10 mg) was added as an antioxidant. Extraction was followed by homogenization in a chilled blender for 10 minutes. The homogenate was allowed to stand overnight (at 4°C) in the dark, filtered, and the residue was reextracted. The filtrate was evaporated (at 35°C) in a rotary evaporator, avoiding direct sunlight, and an aqueous extract remained.
The method of manufacture of Pyrus Malus (Apple) Seed Extract that is being marketed to the cosmetics industry has been described as follows: 25 Ground apple seeds are extracted with alcohol (95% non-denatured alcohol). The solvent is evaporated to remove any residual alcohol. The residue is a paste that is sold in solution of various solvents.
Composition/Impurities
The composition of various components of the apple tree will vary based on where the tree is grown, the maturity of the tree, and storage conditions for the parts of the tree that are used to produce cosmetic ingredients. The method of extraction will also affect the composition of ingredients derived from the parts of the apple tree.
Pyrus Malus (Apple) Bark Extract
According to information provided by the cosmetics industry, Pyrus Malus (Apple) Root Bark Powder extracted with carbon dioxide is purified to contain 95% to 98% phloridzin, a dihydrochalcone.
22
According to another source, the following 3 compounds were isolated from the bark of
Malus Domestica (Apple) Flower Extract
The extract of flowers from the redgold apple cultivar was analyzed using gas chromatography (GC) and GC-MS (mass spectrometry), and the following compounds identified were classified as making a major contribution to the odor of apple flowers: 26
benzyl alcohol
2-phenylethyl alcohol
cinnamyl alcohol
nonanal
octanol
indole
Pyrus Malus (Apple) Fruit
Composition Data on Different Varieties of
Mineral Composition/Chemistry Data on Different Varieties of
A number of sweet and sour varieties of apples from the hilly parts of Northwest India have been found to contain malvidin monoglycoside. 28 Additionally, the skin of Grimes Golden, Jonathon, and Stayman wine sap apples have been reported to contain idaein (3-β-galactosidyl cyanidin), and the related yellow varieties yielded quercetin-3-galactoside.
The following 3 predominant hydroxycinnamic derivatives in the apple (
Assay results for the presence of amino acids in
Pyrus Malus (Apple) Fiber
Malus Domestica (Apple) Fruit and Malus Domestica (Apple) Peel
For 20 apple cultivars, the concentrations of 5 classes of polyphenols were significantly different, across cultivar, for both the peel and flesh. 33 The total polyphenol concentration ranged from .9 µg/g wwb (wet weight basis) in the flesh of Newtown Pippin to 453 µg/g wwb in the peel of Red Delicious. Harrison, Granny Smith, Rome, Winesap, and Black Twig cultivars contained the highest concentration of total flavan-3-ols in flesh.
Apple Fruit (genus and species not stated)
Data on the average content of phenolic compounds in fruit from apple trees (genus and species not stated; semi-dwarf and super-dwarf rootstocks) grown in Lithuania are as follows:
34
Whether or not the fruit was ripe was not stated. • Chlorogenic acid (729-1047 µg/g dry weight) • Phloridzin (83.7-122 µg/g dry weight) • Procyanidin B1 (33.5-81.5 µg/g dry weight) • Procyanidin B2 (504-920 µg/g dry weight) • Σ (Σ = total) Procyanidins (558-1001 µg/g dry weight) • (−)-Epicatechin (217-329 µg/g dry weight) • Σ Catechins (254-406 µg/g dry weight) • Hyperoside (87-147 µg/g dry weight • Isoquercitrin (15.4-23.3 µg/g dry weight) • Rutin (15.1-21.6 µg/g dry weight) • Avicularin (59.9-94 µg/g dry weight) • Quercitrin (86.1-130 µg/g dry weight) • Σ Quercetin glycosides (264-416 µg/g dry weight) • Σ Phenolic compounds (1976-2943 µg/g dry weight)
Super-dwarf rootstocks had the highest content of all phenolic compounds tested, and semi-dwarf rootstocks had the lower content of all phenolic compounds tested. The content of (+)-catechin, procyanidin B1, and total procyanidins in apple fruits depended on the rootstock genotype. The rootstock genotype had a lesser effect on the content of quercitrin, (−)-epicatechin, total catechins, phloridzin, and chlorogenic acid. It was noted that conditions during the growing season, yield, and fruit weight had an impact on the content of phenolic compounds.
Pyrus Malus (Apple) Fruit Extract
Using paper chromatography, the extracts (extractant not stated) of fruits of
According to information provided by the cosmetics industry, • Quercetin • Mineral substances • Essential oil • Enzymes • Fruit acids • Amino acids • Vitamins • Arabane • Galactane • Tannins • Pectin • Sugars • Wax One trade name mixture contains Pyrus Malus (Apple) Fruit Extract (10 - 25%) and propylene glycol (75 - 100%),
6
and the other contains Pyrus Malus (Apple) Fruit Extract (10 - 25%), glycerin (75 - 100%), and aqua (water) (10 - 25%).
7
The composition (polyphenol profiles) of a Pyrus Malus (Apple) Fruit Extract trade name material (identified as apple polyphenol extract) has been described as follows: 17 procyanidins (63.8%), which comprised 11.1% dimers, 12.3% trimers, 8.7% tetramers, 5.9% pentamers, 4.9% hexamers, and 20.9% other polymers. It also contained 12.4% flavan-3-ols (monomers), 6.5% other flavonoids, 10.8% non-flavonoids. In addition to the polyphenols, apple polyphenol extract also contained 1.8% moisture, 2.1% protein, and .4% ash.
Pyrus Malus (Apple) Juice
Juice from the Fuji and Gala apple varieties (genus and species not stated) in Brazil has the following composition: 19 malic acid (.18 - .389 g/100 mL), total reducing sugar (8.65 - 15.18 g/100 mL), and total phenolic compounds (100 - 400 mg/L). In comparison, apple juice from the Golden delicious variety contains: malic acid (.312 g/100 mL), total reducing sugar (10.533 g/100 mL), and total phenolic compounds (535.082 mg/L).
Pyrus Malus (Apple) Leaf and Malus Domestica (Apple) Leaf Extract
The partition of nitrogen in various parts of
A study relating to the composition and content of phenolic compounds in ethanol extracts of apple (
hyperoside
isoquercitrin
avicularin
rutin
quercitrin (major compound among the quercetin glycosides identified)
Using an ultra-performance liquid chromatography-diode array detection (UPLC-DAD) quantification method, the following 4 compounds were identified in apple leaves of two different varieties, golden and royal: 38
rutin
3-hydroxyphloridzin
phloridzin
quercetin-3-
The volatile oil obtained from fresh leaves of the
Pyrus Malus (Apple) Pectin Extract
In the absence of data on the extract, it should be noted that pectin is a complex mixture of polysaccharides that comprises approximately one third of the cell wall dry substance of higher plants. 20 The highest concentrations of pectin are found in the middle lamella of the cell wall, with concentrations gradually decreasing from the primary wall toward the plasma membrane. The Panel has reviewed the safety of pectin in cosmetics and concluded that this ingredient is safe in the present practices of use and concentration. 40
Malus Domestica (Apple) Peel Extract
The total phenolic and anthocyanin content of apple peels from 6 apple (
In another study, variations in the content of phenolics, antioxidant activity, and minerals in the peel and pulp of 5 apple (
Pyrus Malus (Apple) Seed Extract
Endogenous levels of both cis- and trans-isomers of free and bound abscisic acid were studied in dormant and after-ripened
The extracts prepared from dry, dormant
Residual level of pesticides in Pyrus Malus (Apple) Seed Extract is controlled to comply with European food limits. 25
Apple Seed Extract
Fatty Acid Composition Data on Apple Seed Extract. 44
Further extraction of the seed with 70% aqueous acetone yielded the following 2 major compounds: [(6-
Use
Cosmetic
The safety of Pyrus malus (apple)-derived ingredients is evaluated based on the expected use of these ingredients in cosmetics. The Panel uses data received from the US FDA and the cosmetics industry to determine cosmetic use. Use frequencies of individual ingredients in cosmetics are collected from manufacturers and reported by cosmetic product category in the FDA VCRP database. Use concentration data are submitted by the cosmetics industry in response to surveys of maximum reported use concentrations, by product category, that are conducted by the Council. Collectively, the use frequency and use concentration data indicate that 16 of the 26 apple-derived ingredients are used in cosmetic products.
According to information supplied by the VCRP and the results from a survey of ingredient use concentrations conducted by the Council, 9
Pyrus Malus (Apple) Bark Extract
Pyrus Malus (Apple) Carpel Powder
Pyrus malus (Apple) Fiber
Pyrus Malus (Apple) Leaf Extract
Pyrus Malus (Apple) Peel Extract
Pyrus Malus (Apple) Peel Wax
Pyrus Malus (Apple) Pulp Extract
Pyrus Malus (Apple) Root Bark Powder
Pyrus Malus (Apple) Stem Extract
NR = not reported; Totals = rinse-off + leave-on product uses.
aIt is possible that these products may be sprays, but it is not specified whether the reported uses are sprays.
bIt is possible that these products may be powders, but it is not specified whether the reported uses are powders.
cNot specified whether a powder or spray, so this information is captured for both categories of incidental inhalation.
Cosmetic products containing
The following ingredients are being used in products that may be inhaled: Pyrus Malus (Apple) Fruit Extract, pyrus malus (apple) flower extract, Pyrus Malus (Apple) Fruit, Malus Domestica (Apple) Fruit Extract, and Malus Domestica (Apple) Fruit Water. The highest maximum use concentration that is being reported for
Noncosmetic
Apples are among the 20 most frequently consumed raw fruits in the United States. 50
Toxicokinetics
Apple Juice
Unfiltered apple juice (500 mL) was consumed by human subjects, and blood and urine samples were analyzed for total phenolic content and the concentration of selected individual polyphenolic compounds. 51 Large differences in apple polyphenol pharmacokinetics among subjects were observed. Pharmacokinetic data were classified into subgroups according to fast or slow rates of polyphenol metabolism. For some of the subjects, metabolism was undetectable within the time frame of the study. No differences in renal excretion were detected when male and female subjects were compared. However, relative concentrations of polyphenolic compounds were slightly higher in male subjects. Thus, apple-derived polyphenols can be readily detected in human blood and urine after apple juice consumption. The authors noted that the existence of subpopulations with different pharmacokinetics was suggestive of significant variations in individual metabolism rates of polyphenolic substances.
Toxicology
As noted earlier, some of the ingredients (e.g., fruit/fruit-derived) reviewed in this safety assessment may be consumed as food, and daily exposure from food use would result in much larger systemic exposures than those from use in cosmetic products. The primary focus of the safety assessment of these ingredients as used in cosmetics is on the potential for local effects from topical exposure.
Toxicity data on procyanidin B-2 (epicatechin-(4β→8)-epicatechin, found in apple fruit) were identified in the published literature, and are included for use in the safety assessment of apple fruit extract. As mentioned previously, procyanidins are members of the procyanidin or condensed tannins class of flavonoids. 10 Procyanidin B-2 is not a cosmetic ingredient.
Acute Toxicity
Oral
Apple Fruit Extract
Apple polyphenol extract (dissolved in .5% sodium carboxymethyl cellulose solution) was administered intragastrically to Sprague-Dawley (Crj:CD) rats (5 males, 5 females) at an oral dose of 2000 mg/kg body weight (dose volume = 10 mL/kg). 17 The production of apple polyphenol extract is described in the Method of Manufacture section of this safety assessment. The animals were necropsied after a 14-d observation period. None of the animals died, and the authors noted that there were no significant changes in organs examined. It was concluded that the acute oral minimum fatal dose of apple polyphenol extract was >2000 mg/kg body weight.
Pyrus Malus (Apple) Seed Extract
The acute oral toxicity of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was studied using 10 male mice (IFFA CREDO OF 1). 25 The test substance was administered at a maximal oral dose of 20 mL/kg, and dosing was followed by an 8-d observation period. Gross necropsy was performed on day 8. There was no evidence of test substance-related clinical signs in the study, and the behavior of animals was considered normal. Gross necropsy did not reveal any evidence of modification of the main organs. In particular, there were no signs of necrosis or ulceration of the digestive tract.
Subcutaneous
Procyanidin B-2
Procyanidin B-2 (> 94% pure) was administered to groups of ten 6-wk-old Sprague-Dawley rats of the Crj: CD, SPF/VAF strain (5 males, 5 females/group).9,10 The test material (in purified water) was administered subcutaneously to 3 groups at doses of 500 mg/kg, 1000 mg/kg, and 2000 mg/kg, respectively (dose volume = 10 mL/kg). The single dose was injected into the neck. A fourth group (control) was dosed with purified water. None of the animals died. Hair loss and crust formation (in all 3 dose groups), and swelling (2000 mg/kg group) were reported. The following observations were made at necropsy on day 14: thickening of the subcutis with granulomatous inflammation in the 1000 mg/kg group (4 of 5 males; 2 of 5 females) and 2000 mg/kg group (all animals); accumulation of pigment-laden macrophages in the duodenal mucosa in the 500 mg/kg group (1 of 5 females), 1000 mg/kg group (4 of 5 males; all females), and 2000 mg/kg group (all animals).
Repeated Dose Toxicity
Oral
Apple Fruit Extract
Apple polyphenol extract was administered intragastrically to 3 groups of Sprague-Dawley (Crj:CD) rats (10 males, 10 females) at doses of 500; 1000; and 2000 mg/kg/d (dose volume = 10 mL/kg). 17 The doses were administered daily for 90 d. A fourth group served as the untreated control. Necropsies were performed on all animals at the end of the study. Body weight gain was unaffected by treatment, and changes in food consumption were attributed to normal biological variation. Statistically significant differences (P < .05 or P < .01) in hematological, clinical chemistry, and urinary tests were reported. However, these changes were within the normal range of physiological background data and were not correlated with the apple polyphenol extract dosage. Gross necropsy findings were not indicative of adverse changes in any of the organs examined. A statistically significant increase in lung weight was found in the 1000 mg/kg/d dose group, but this change was within the normal physiological range. Histopathological examination of organs did not reveal any changes that were related to dosing with the test material.
Antimicrobial Activity
Pyrus Malus (Apple) Pectin Extract
Pectin was extracted from several varieties of apple, i.e., American, Delicious, and Maharaj-ji (all obtained from Kashmir).
20
Pectin content was found to be maximum in Maharaj-ji (20.60%), followed by Delicious (14.4%) and American (11.60%). The pectin extracted was evaluated for in vitro antibacterial activity against different pathogenic bacterial cultures and antifungal activity. Pectin extracted from the Delicious variety had potent antibacterial activity against
Reproductive and Developmental Toxicity
Data on the reproductive and developmental toxicity of apple-derived ingredients were not found in the published literature nor were unpublished data provided.
Genotoxicity
In Vitro
Apple Fruit Extract
The genotoxicity of apple fruit extract was evaluated in the Ames test using
Apple fruit extract (concentrations up to .313 mg/mL) was evaluated for genotoxic activity in Chinese hamster CHL/IU mammalian cells using a chromosomal aberrations assay with and without metabolic activation. 10 Genotoxicity was not observed, with or without metabolic activation.
Pyrus Malus (Apple) Seed Extract
The genotoxicity of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was studied using the following
Procyanidin B-2
The genotoxicity of procyanidin B-2 (> 94% pure) was evaluated using
In the chromosome aberrations assay using Chinese hamster lung cells, procyanidin B-2 (> 94% pure) was evaluated with (concentrations up to 4.8 mM) and without (concentrations up to 1.8 mM) metabolic activation. 9 Neither structural aberrations such as chromosome-type aberrations or chromatid-type aberrations were observed with or without metabolic activation. However, polyploidy was observed with metabolic activation.
In Vivo
Apple Fruit Extract
The genotoxicity of apple fruit extract was evaluated in the micronucelus test. 17 Three groups of 5 male Sprague-Dawley rats (7-wk-old) were dosed orally with 500 mg/kg body weight; 1000 mg/kg body weight; and 2000 mg/kg body weight (dose volume = 10 mL/kg), respectively. Genotoxicity was evaluated by measuring the frequency of polychromatic erythrocytes cells in bone marrow. Test results were negative.
Procyanidin B-2
In a micronucleus test, groups of 8-wk-old mice [Crj: CD-1 (ICR), specific pathogen free (SPF)/virus antibody free (VAF)] received single subcutaneous injections of 500 mg/kg, 1000 mg/kg, and 2000 mg/kg procyanidin B-2, respectively. 9 The frequency of micronucleated polychromatic erythrocytes in the bone marrow of mice dosed with procyanidin B-2 was not significantly different from that of the negative control.
Carcinogenicity
Data on the carcinogenicity of apple-derived ingredients were not found in the published literature nor were unpublished data provided.
Anticarcinogenicity
The anticarcinogenicity of apples and their components has been the subject of a review article. 52 In in vitro studies, apple extracts and components, especially oligomeric procyanidins, have been shown to influence multiple mechanisms that are relevant for cancer prevention. Additionally, apple products have been shown to prevent skin, mammary, and colon carcinogenesis in animal models, and epidemiological observations indicate that regular consumption of one or more apples a day may reduce the risk for lung and colon cancer.
Irritation and Sensitzation
Skin Irritation and Sensitization
In Vitro
Pyrus Malus (Apple) Fruit Extract
The skin irritation potential of a tradename material containing 20% Pyrus Malus (Apple) Fruit Extract was evaluated using the EpidermTM model, a reconstructed human epidermal model consisting of human-derived epidermal keratinocytes. 53 This is a European Center for the Validation of Alternative Methods (ECVAM)-validated test method. 54 The keratinocytes were cultured to form a multilayer, highly differentiated model of the human epidermis. In the EpidermTM model assay, a chemical is classified as an irritant if the mean relative tissue viability of exposed tissues is reduced by 50% of the mean viability of the negative controls. A non-irritant’s viability is >50%. Sterile Dulbecco’s phosphate-buffered saline (DPBS) and sterile deionized water served as negative controls. Sodium dodecyl sulfate solution (5%) served as the positive control. The test material was classified as a non-irritant in this assay. The positive control was classified as an irritant. 53 In the same assay, negative results were also reported for another tradename material containing 20% Pyrus Malus (Apple) Fruit Extract. 55
Non-Human
Pyrus Malus (Apple) Fruit Extract
A product described as an aqueous solution containing 20% Pyrus Malus (Apple) Fruit Extract was applied for 4 h (under a dressing [type not stated]) to the skin of 3 albino rabbits according to the Organization for Economic Co-operation and Development (OECD) Test Guideline (TG) 404. 16 Test sites were examined for up to 72 h after removal of the dressing. The test substance was classified as having slight skin irritation potential.
The skin sensitization potential of a product described as an aqueous solution containing 20% Pyrus Malus (Apple) Fruit Extract was studied in the maximization test (OECD TG 406) using albino guinea pigs (number not stated). 16 Positive reactions were not observed. However, it was concluded that the test substance had slight skin sensitization potential, but did not induce skin irritation.
Pyrus Malus (Apple) Seed Extract
The skin irritation potential of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was evaluated using 6 New Zealand white rabbits. 25 Prior to application, the test substance was diluted to a concentration of 23% in sterile distilled water (effective test substance concentration = 1% × 23% = .23%). An occlusive patch containing the diluted test substance (.5 mL) was applied for 24 h to abraded and intact skin on the right and left side, respectively. The dose per cm2 was not stated. Reactions were scored according to the Draize scale at 24 h, 48 h, and 72 h post-application. Mild erythema was observed at intact and abraded test sites of all animals, but was completely reversible between 48 h and 72 h post-application. Edema was not observed. The test substance was classified as a slight skin irritant (index of cutaneous irritation = .58).
The guinea pig maximization test was used to evaluate the skin sensitization potential of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) in 20 male guinea pigs (strain not stated). 25 During induction, the animals were injected intradermally with the test substance, followed by topical dermal application of the test substance (25% in petroleum jelly) under an occlusive patch for 48 h. The effective concentration of the test substance applied dermally during induction was .25% (1 % × 25 % = .25%). The dose per cm2 was not stated. During the challenge phase, the test substance (.25%) was applied to the flank, under an occlusive, patch for 24 h. An additional 20 guinea pigs served as controls (treated similarly, but without test substance application). Reactions were scored at 24 h and 48 h post-application. Mild erythema was observed in treated and control animals, and reactions had cleared by 48 h. The erythema observed was interpreted as a consequence of an irritation phenomenon, rather than as a sensitization phenomenon. This was so because the erythema observed at 24 h was reported for an equal number of treated and control animals, and because the percentage of the reaction observed was extremely low. The test substance was classified as a non-sensitizer.
Procyanidin B-2
Procyanidin B-2 (vehicle not stated) was applied topically to dorsal abraded and intact skin of 12 New Zealand white rabbits (Kbs: NZW, SPF) using an occlusive patch (.5 mL on 2.5 × 2.5 cm patch). 9 Sodium lauryl sulfate (8% aqueous) and physiological saline served as positive and negative controls, respectively. The patches remained in place for 24 h, and reactions were scored at 24.5 h and 72 h post-application. Skin irritation was not observed at abraded or intact sites treated with procyanidin B-2, the vehicle, or saline. Significant inflammation (severe irritation) was observed at sites treated with 8% sodium lauryl sulfate.
The skin sensitization potential of procyanidin B-2 was evaluated in a maximization test using 24 male guinea pigs (Crj: Hartley,SPF/VAF). 9 A 2 × 4 cm2 area of dorsal skin was used in the induction phase of the study, and the test procedure involved a combination of subcutaneous injections and topical applications. During the induction phase, procyanidin B-2 (1%) was injected subcutaneously, and, after 24 h, an occlusive patch containing .2 mL of 10% procyanidin B-2 was applied for 24 h. The challenge phase was initiated after a 2-wk non-treatment period. A 2 × 2 cm2 occlusive patch containing 10% procyanidin B-2 (.1 mL) was applied for 24 h. Reactions were scored at 24.5, 48, and 72 h post-application. 2,4-Dinitrochlorobenzene (1% DNCB) served as the positive control. Procyanidin B-2 did not induce sensitization. DNCB was a strong sensitizer.
Human
Pyrus Malus (Apple) Fruit Water
The skin irritation and sensitization potential of a face and neck product containing 8.78% Pyrus Malus (Apple) Fruit Water was evaluated in a human repeated insult patch test involving 49 male and female subjects. 56 During induction, an occlusive patch (2 cm2) containing the test substance (.2 mL) was applied for 24 h to an area between the scapula and waist that was adjacent to the midline. A total of 9 induction applications were made. A 2-wk non-treatment period was followed by the challenge phase. A challenge patch was applied for 24 h to a new test site. Reactions were scored at 24 h and 72 h post-application. Transient, barely perceptible (+) to mild (1-level) reactions (all non-specific patch test responses) were observed in 5 or 49 subjects during the induction and/or challenge phase of the study. It was concluded that the test substance did not induce skin irritation or cause allergic contact dermatitis.
Phototoxicity
Pyrus Malus (Apple) Seed Extract
The phototoxicity of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was studied using albino guinea pigs. 25 The animals (10 per group) received an application of the test substance (1 mL) with or without long-wave ultraviolet light (UVA) irradiation. The dose per cm2 was not stated. Test sites were examined at 1, 6, and 24 h post-irradiation. There was no evidence of dermal reactions in irradiated or non-irradiated animals, and the test substance was classified as non-phototoxic.
Ocular Irritation
In Vitro
Pyrus Malus (Apple) Fruit Extract
The ocular irritation potential of a tradename material containing 20% Pyrus Malus (Apple) Fruit Extract was evaluated using the EpiocularTM model, a multilayer, highly differentiated model of the human corneal epithelium. 53 In the EpiocularTM model assay, a chemical is classified as an irritant if the mean relative tissue viability of the exposed tissues is reduced by 60% of the mean viability of the negative controls. A non-irritant’s viability is >40%. Sterile DPBS and sterile deionized water served as negative controls. Methyl acetate served as the positive control. The test material was classified as a non-irritant in this assay. The positive control was classified as an irritant. In the same assay, negative results were also reported for another tradename material containing 20% Pyrus Malus (Apple) Fruit Extract. 55
Using the neutral red uptake assay, the ocular irritation potential of a product described as an aqueous solution containing a maximum percentage of 20% Pyrus Malus (Apple) Fruit Extract was determined using a monolayer of rabbit corneal fibroblasts. 16 The product was tested at concentrations up to 50% (effective test substance concentration = 20% × 50% = 10%) In this assay, cytotoxicity was evaluated by determining the test concentration that caused 50% cell death (IC50). Test results indicated that the IC50 was above a concentration of 50%. The % cell death that was observed at a dilution of 50% was 15%, and, thus, cytotoxicity was said to have been negligible.
In Vivo
Pyrus Malus (Apple) Fruit Extract
A product described as an aqueous solution containing a maximum percentage of 20% Pyrus Malus (Apple) Fruit Extract (.1 mL) was instilled into one eye of each of 3 rabbits according to the OECD TG 405. 16 Instillation was not followed by ocular rinsing. Untreated eyes served as controls. The test substance was slightly irritating to the eye and did not cause lesions of the ocular mucosa that were considered significant.
Pyrus Malus (Apple) Seed Extract
Pyrus Malus (Apple) Seed Extract (.1 mL; 1% in hexyldecanol) was instilled into the right eye of each of 6 New Zealand white rabbits, and remained for 24 h. 25 Ocular irritation was scored according to the method of Kay and Calandra at 12, 24, 48, 72, and 96 h post-instillation. At 24 h post-instillation, conjunctival redness, swelling, and discharge were observed in 6 rabbits. These reactions were completely reversible at 48 h and 96 h post-instillation. Lesions of the cornea or iris were not observed. The test substance was classified as slightly irritating to the eyes (accurate ocular irritation index at 24 h = .58).
Procyanidin B-2
The ocular irritation potential of procyanidin B-2 (> 94% pure) was evaluated using twelve male New Zealand white rabbits (Kbs: NZW, SPF). 9 The test material (100 µl) was instilled in the right eye of each animal. The left eye served as the untreated control. Half of the animals were subjected to ocular rinsing after instillation. Ocular reactions were scored for up to 120 h post-instillation. No changes were observed in the cornea or iris, but slight irritation of the conjunctiva was observed. Procyanidin B-2 was classified as minimally irritating to the eyes of rabbits.
Miscellaneous Studies
Phloridzin
A study on phloridzin-induced melanogenesis was performed using B16 melanoma cells. 57 Phloridzin (found in many parts of the apple tree) induced a dose-dependent increase (100 µg/ml and 500 µg/ml doses) in tyrosinase activity and melanin content, and these changes were accompanied by an increase in the levels of tyrosinase and the tyrosine-related proteins, TRP-1 and TRP-2. Also, the cAMP-dependent protein kinase A (PKA) inhibitor H89 impaired the response of the tyrosinase activity and melanin synthesis to phloridzin. Collectively, the results of this study indicated that phloridzin increased tyrosinase gene expression through the cAMP signaling pathway, leading to stimulation of melanogenesis.
Summary
According to information supplied by the VCRP and the results from a survey of ingredient use concentrations conducted by the Council, the following 9 Pyrus Malus (Apple) Fruit Extract Pyrus malus (Apple) Flower Extract Pyrus Malus (Apple) Fruit Pyrus Malus (Apple) Fruit Water Pyrus Malus (Apple) Juice Pyrus Malus (Apple) Pectin Extract Pyrus Malus (Apple) Peel Powder Pyrus Malus (Apple) Root Extract Pyrus Malus (Apple) Seed Extract Malus Domestica (Apple) Fruit Extract Malus Domestica (Apple) Fruit Water Malus Domestica (Apple) Fiber Malus Domestica (Apple) Juice Malus Domestica (Apple) Oil Malus Domestica (Apple) Stem Extract Malus Domestica (Apple) Fruit Cell Culture Extract
The available information on the composition of apples indicates that phenolic compounds are present in the fruit, juice, leaves, peel, and seeds. Phloridzin (found in many parts of the apple tree) induced a dose-dependent increase in tyrosinase activity and melanin content in B16 melanoma cells, and these changes were accompanied by an increase in the levels of tyrosinase and the tyrosine-related proteins, TRP-1 and TRP-2.
Apple-derived polyphenols were readily detected in human blood and urine after apple juice consumption.
In an acute oral toxicity study of Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol), there was no evidence of test substance-related clinical signs. At gross necropsy, there were no signs of necrosis or ulceration of the digestive tract, or modification of the main organs. In acute oral and repeated dose toxicity tests (rats) on apple fruit extract, there were no significant hematological, clinical, chemical, histopathological, or urinary effects at a dose of 2000 mg/kg.
A single subcutaneous injection of procyanidin B-2 into rats did not produce signs of significant injury, and a lethal dose of >2000 mg/kg was reported.
In the Ames test without metabolic activation, apple fruit extract was slightly genotoxic when tested at a high concentration of 2500 µg/plate, but significant genotoxic activity was not found in the chromosomal aberration test or the micronucleus test. In another Ames test, Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was non-genotoxic in the following
An aqueous solution containing 20% Pyrus Malus (Apple) Fruit Extract was slightly irritating to the skin of rabbits. In the guinea pig maximization test, this solution did not induce skin irritation, but had slight skin sensitization potential. The skin irritation potential of a tradename material containing 20% Pyrus Malus (Apple) Fruit Extract was evaluated using the EpidermTM model, and results were negative.
Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) caused slight skin irritation when diluted with water to a concentration of .23% and applied to the skin of rabbits. In the guinea pig maximization test, Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) caused skin irritation, but not sensitization, when tested at an ingredient concentration of .25% during induction and challenge. A preparation containing procyanidin B-2 did not cause skin irritation in rabbits. Additionally, procyanidin B-2 did not induce skin sensitization in guinea pigs in the maximization test.
A face and neck product containing 8.78% Pyrus Malus (Apple) Fruit Water did not cause skin irritation or allergic contact dermatitis in an RIPT involving 49 subjects.
Pyrus Malus (Apple) Seed Extract (1% w/v in hexyldecanol) was classified as non-phototoxic in a study involving albino guinea pigs.
An aqueous solution containing a maximum percentage of 20% Pyrus Malus (Apple) Fruit Extract (.1 mL) was slightly irritating to the eyes of rabbits. The same was true after instillation of Pyrus Malus (Apple) Seed Extract (1% in hexyldecanol) into the eyes of rabbits. In primary ocular irritation tests involving rabbits, both a preparation containing procyanidin B-2 and the vehicle induced slight conjunctival irritation; however, ethanol was presumed to have been the causative agent.
The ocular irritation potential of an aqueous solution containing a maximum percentage of 20% Pyrus Malus (Apple) Fruit Extract, tested at ingredient concentrations up to 10%, was evaluated using the method of neutral red release. Cytotoxicity was evaluated in this assay and found to be negligible; thus, ocular irritation was negligible. A tradename material containing 20% Pyrus Malus (Apple) Fruit Extract was evaluated for ocular irritation potential using the EpiocularTM model, and results were negative.
Discussion
The Panel discussed different apple cultivars as sources of apple-derived ingredients, the stimulatory effect of phloridzin (component of apple fruit) on melanogenesis, the presence of impurities in botanical ingredients, and the potential for inhalation exposure to apple-derived cosmetic ingredients. The data needed for completion of the safety assessment on 5 of the apple-derived ingredients were also determined.
Considering that apple-derived ingredients may be obtained from different apple cultivars, the Panel noted that the composition of ingredients derived from different cultivars should be similar to the composition of ingredients reviewed in this safety assessment.
The Panel stated their awareness of data indicating that phloridzin, a component of apple fruit, apple bark extract, apple root bark powder, apple leaf extract, and apple seed extract, had a stimulatory effect on melanogenesis in vitro, but agreed that a review of ingredients for drug effects, i.e., noncosmetic effects, is not within the Panel’s purview. However, the Panel noted that an effect on cutaneous pigmentation would not be expected at the use concentrations of apple-derived ingredients in cosmetic products. As a precaution, the Panel noted that manufacturers should be aware of this effect and, thus, the ingredients containing phloridzin in cosmetic formulations should not have a stimulatory effect on melanogenesis when applied to the skin.
The Panel expressed concern about pesticide residues and heavy metals that may be present in botanical ingredients. They stressed that the cosmetics industry should continue to use current good manufacturing practices (cGMPs) to limit impurities. The Panel noted the USDA designation of ≤15 ppb as corresponding to “negative” aflatoxin content. Additionally, the Panel stated that aflatoxins should not be present at levels of toxicological concern in apple-derived ingredients.
Additionally, the Panel discussed the potential for incidental inhalation exposures to apple-derived ingredients in products that are sprayed or in powder form and agreed that, based on likely airborne particle size distributions and concentrations in the breathing zone and ingredient use, incidental inhalation would not lead to local respiratory effects or systemic effects.
The Panel determined that the following data are needed to evaluate the safety of Pyrus Malus (Apple) Root Extract, Pyrus Malus (Apple) Stem Extract, Malus Domestica (Apple) Callus Extract, Malus Domestica (Apple) Oil, and Malus Domestica (Apple) Stem Extract in cosmetic products: • Method of manufacture and impurities • 28-day dermal toxicity study; if any adverse effects noted, genotoxicity and reproductive and developmental toxicity data may be needed
Conclusion
The Expert Panel for Cosmetic Ingredient Safety concluded that the following 21 apple-derived ingredients are safe in the present practices of use and concentration in cosmetics as described in this safety assessment, when formulated to be non-irritating and non-sensitizing. Pyrus Malus (Apple) Bark Extract* Pyrus Malus (Apple) Carpel Powder* Pyrus Malus (Apple) Fiber* Pyrus Malus (Apple) Flower Extract Pyrus Malus (Apple) Fruit Extract Pyrus Malus (Apple) Fruit Pyrus Malus (Apple) Fruit Water Pyrus Malus (Apple) Juice Pyrus Malus (Apple) Leaf Extract* Pyrus Malus (Apple) Pectin Extract Pyrus Malus (Apple) Peel Extract* Pyrus Malus (Apple) Peel Powder Pyrus Malus (Apple) peel Wax* Pyrus Malus (Apple) Pulp Extract* Pyrus Malus (Apple) Root Bark Powder* Pyrus Malus (Apple) Seed Extract Malus Domestica (Apple) Fiber Malus Domestica (Apple) Fruit Extract Malus Domestica (Apple) Fruit Water Malus Domestica (Apple) Fruit Cell Culture Extract Malus Domestica (Apple) Juice
*Not reported to be in current use. Were ingredients in this group not in current use to be used in the future, the expectation is that they would be used in product categories and at concentrations comparable to others in this group.
The Panel concluded that the available data are insufficient for evaluating the safety of the following 5 apple-derived ingredients in cosmetic products: Pyrus Malus (Apple) Root Extract Pyrus Malus (Apple) Stem Extract Malus Domestica (Apple) Callus Extract Malus Domestica (Apple) Oil Malus Domestica (Apple) Stem Extract