Safety Assessment of

Silk Protein Ingredients as Used in Cosmetics

Status: Final Report

Release Date: February 8, 2016
Panel Date: December 14-15, 2015

The 2016 Cosmetic Ingredient Review Expert Panel members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V.
Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; James G. Marks, Jr., M.D.; Ronald
C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is Lillian J. Gill, D.P.A.
This report was prepared by Wilbur Johnson, Jr., M.S., Senior Scientific Analyst.

© Cosmetic Ingredient Review

1620 L STREET, NW, SUITE 1200 ◊ WASHINGTON, DC 20036-4702 ◊ PH 202.331.0651 ◊ FAX 202.331.0088 ◊ CIRINFO@CIR-SAFETY.ORG
Abstract: The Cosmetic Ingredient Review (CIR) Expert Panel (Panel) reviewed the safety of hydrolyzed silk and 9 other
silk protein ingredients, which function primarily as skin and hair conditioning agents and bulking agents in cosmetic
products. The Panel reviewed relevant data relating to the safety of these ingredients and concluded that 8 ingredients are
safe in the present practices of use and concentration in cosmetics, as described in this safety assessment, but that the
available data are insufficient for determining the safety of two silk protein ingredients in cosmetic products, MEA-
hydrolyzed silk and silkworm cocoon extract.

INTRODUCTION

The safety of the following 10 silk protein ingredients as used in cosmetics is reviewed in this safety assessment:

Fibroin Sericin
Hydrolyzed Fibroin Silk
Hydrolyzed Sericin Silk Extract
Hydrolyzed Silk Silk Powder
MEA-Hydrolyzed Silk Silkworm Cocoon Extract

According to the International Cosmetic Ingredient Dictionary and Handbook, these ingredients are reported to function as
skin and hair conditioning agents and bulking agents in cosmetic products.1

Silk is a fibrous protein that is a product of the silk worm, Bombyx mori, and spiders (Nephila clavipes and Araneus
diadematus). Silk proteins are usually produced within specialized glands. These proteins are biosynthesized in epithelial
cells and secreted into the lumen of these glands, where the proteins are stored prior to being spun into silk fibers.2 The silk
worm (Bombyx mori) is the source of silk from which cosmetic ingredients are derived.1

MEA-hydrolyzed silk is evaluated in this safety assessment, and it should be noted that the Panel has issued a final
amended report with the conclusion that the following ingredients are safe in the present practices of use and concentration
described in the report (rinse-off products only) when formulated to be non-irritating:3 ethanolamine MEA, ethanolamine
HCl, MEA-benzoate, MEA-cocoate, MEA-laureth-6 carboxylate, MEA-laureth sulfate, MEA-lauryl sulfate, MEA-PPG-6
laureth-7 carboxylate, MEA-PPG laureth-6 carboxylate, MEA-PPG-8 steareth-7 carboxylate, MEA-salicylate, MEA-sulfite,
MEA-tallowate, and MEA-undecylenate. The Panel also cautioned that these ingredients should not be used in cosmetic
products in which N-nitroso compounds may be formed.

Data on skin depigmentation effects are included in this safety assessment; however, skin depigmentation is
considered a drug and not a cosmetic effect in the United States, and is not within the Panel’s purview.

CHEMISTRY

Definition and Structure

The silkworm, Bombyx mori, produces silk proteins during the final stage of larval development, and two silk
proteins, fibroin and sericin, have been distinguished as major components of silk cocoons.4 All of the ingredients in this
report are related because they are derived from the silk fibers produced by Bombyx mori. The definitions and functions of
fibroin, sericin, and other silk protein ingredients reviewed in this safety assessment are presented in Table 1.1

Fibroin

Bombyx mori (B. mori) silk fibroin was determined to exist as a repeated type II β-turn structure, wherein the
conformation of one chain enables the formation of intra-molecular hydrogen bonds with two juxtaposed (adjacent) chains.5
Sericin

Circular dichroism and infrared absorption spectra show that the molecular configuration of sericin is mainly
random crimp.6,7 X-ray diffraction analysis and differential thermal analysis indicate that the assembled structure or sericin
powder is an amorphous structure;7 however, the amorphous sericin transforms into a β-structure in the presence of water.8

Chemical and Physical Properties

Properties of fibroin, sericin, and other silk protein ingredients, are summarized in Table 2. Polarization microscopy
shows that, in silk, sericin forms three layers surrounding a fibroin fiber.5

Method of Manufacture

Silk

Fibroin (main protein of silk) and sericin (another silk protein) are secreted by insect silk glands. Fibroin, in
aqueous solution, is converted into silk fibers by a process that is called spinning.12,13 According to another source, in the
process of manufacturing lustrous silk from the dried cocoons of silkworms, fibroin is separated from sericin, the other major
component of the cocoon, by a degumming process, and the sericin is mostly discarded in the wastewater.10

Several methods have been reported for removing sericin in the degumming process of cocoons. Practically all
industrial removal processes involve extraction with soaps and detergents. Heat and acid extraction are other methods.
Sericin extracted by different methods can yield different amino acid compositions.10

Additional information indicates that some commercial silk is prepared from natural silk by removing sericin, and
that the purified aqueous fibroin is dried and pulverized into a powder.14

Hydrolyzed Silk

Hydrolyzed silk has been reported to be prepared from the cocoon of the silkworm moth (Bombyx mori).15 The silk
thread is isolated from the cocoon and the fibers are cleaned and degummed. The individual silk fiber is then wound with
other silk fibers to create one long thread. The threads are then combed to remove noils, which are short fibers considered to
be by-products of the textile industry. The noils are used in the production of hydrolyzed silk proteins through carefully
controlled hydrolysis. The resultant material is a 5% solution of a water-soluble silk protein.

It has been reported that hydrolyzed silk (average molecular weight = 300 Da) may be prepared by acid, alkaline, or
enzyme-catalyzed hydrolysis; hydrolyzed silk protein (average molecular weight = 650 Da) may be prepared by alkaline or
enzymatic hydrolysis.16,17 These processes occur for several hours until the desired molecular weight is reached. The final
product is a 20% water solution of hydrolyzed silk protein (mw = 300 Da) or a 6.5% water solution of hydrolyzed silk protein
(mw = 650 Da). Furthermore, another supplier has reported that hydrolyzed silk is prepared by acid and enzyme hydrolysis
until the molecular weight reaches the target range.18

According to other sources, hydrolyzed silk is produced according to the following procedures:19,20,21 Procedure 1:
(1) hydrolysis, (2) inactivation of hydrolytic agent, (3) filtration, (4) treatment, (5) concentration, and (6) sterilization.
Procedure 2: (1) proteins hydrolyzed in water at specific pH and temperature for specific duration, (2) filtration to isolate
desired components, (3) addition of quaternium-15, EDTA, and methylparaben, or just EDTA and methylparaben, and (4)
make batch adjustments if needed (refiltration).

Composition/Impurities

Silk

Silk contains nitrogen (13% to 20%) and, for material from one supplier, the reported or specified maximum
concentration of heavy metals is 20 ppm.14

Hydrolyzed Silk

Data on the composition of hydrolyzed silk are presented in Table 3.22,23 Hydrolyzed silk is marketed as an amino
peptide concentrate that is rich in the 2 proteins that comprise natural silk, sericin and fibroin.24 It consists of ~ 19%
hydrolyzed silk and also contains the preservatives phenoxyethanol (0.4%) and potassium sorbate (0.2%).25 Other
preservatives in hydrolyzed silk include quaternium-15, EDTA, and methylparaben.20,21

Another source indicates that hydrolyzed silk (mw = 300 Da) is marketed as a 20% water solution and that
hydrolyzed silk protein (mw = 650 Da) is marketed as a 6.5% water solution.18 Hydrolyzed silk protein (mw = 300 Da) from
one source was reported to contain heavy metals and arsenic at levels of < 4 ppm and 0.4 ppm, respectively.16 Hydrolyzed
silk protein from another source (mw = 650 Da) was reported to contain heavy metals and arsenic at < 10 ppm and 1 ppm,
respectively.17

According to another supplier, their hydrolyzed silk ingredients are marketed as aqueous solutions, two of which are
20-30% hydrolyzed silk and 27-32% hydrolyzed silk.26

Fibroin

Silk derived from the silkworm Bombyx mori contains two major proteins, fibroin and sericin. Fibroin is a fibrous
protein, present as a delicate twin thread in which the two strands are linked by disulfide bonds and enveloped by successive
sticky layers of sericin.10 Individual filaments are large molecules (3700 amino acids).27 Fibroin has also been described as a
glycoprotein composed of two comparably composed protein subunits covalently linked by disulfide bonds. Fibroin
filaments have both crystalline and amorphous domains.2 The amorphous domains are characterized by the presence of
amino acids with bulkier side chains,28 whereas the crystalline domains are characterized by high percentages of alanine,
glycine and serine.2 Fibroin is a highly insoluble protein containing, as a whole, up to 90% of the amino acids glycine,
alanine and serine.29 According to another source, fibroin contains 46% glycine, 29% alanine, and 12% serine.27

More-detailed information on the composition of fibroin, from the cocoon of the Bombyx mori caterpillar, indicates
that it consists of 2 polypeptide chains, or, more specifically, heavy and light chains of 391 kDa and 25 kDa, respectively; a
disulfide bridge links the heavy chain to the light chain.30 The heavy chains contain 5263 residues, composed of 45.9%
glycine, 30.3% alanine, 12.1% serine, 5.3% tyrosine, 1.8% valine, and only 4.7% of the other 15 amino acid types.

Sericin

Sericin, also referred to as silk glue, is a globular protein that constitutes 25% to 30% of silk proteins. It contains
18 amino acids, most of which have highly polar side-chains, containing hydroxyl, carboxyl or amino groups. The highly
hydrophilic nature of sericin is due to the high content of serine and aspartic acid, approximately 33.4% and 16.7% of sericin,
respectively.10 The predominant amino acids comprising sericin are serine, glycine, and glutamic acid, and side-chain
hydroxyl, carboxyl, and amino groups enable easy cross-linking, copolymerization and blending with other natural or
synthetic polymers.31 According to another source, sericin contains 37% serine, 17% glycine, and 16% aspartate.27

Depending on the solubility, sericin can be separated into three fractions:2 A, B and C. Sericin A (17.2% nitrogen)
comprises the outermost layer and is insoluble in hot water. Sericin B (16.8% nitrogen) is the middle layer and, on acid
hydrolysis, yields the same amino acids as sericin A, and, additionally, tryptophan. Sericin C is the innermost layer,
positioned adjacent to the fibroin strands.

Because the method of production of sericin involves extraction from cocoons using soaps and detergents, alkali
soaps and detergents are typically present as impurities.10

USE

Cosmetic

The safety of the silk protein ingredients included in this safety assessment is evaluated based on the expected use of
these ingredients in cosmetics. The CIR Expert Panel uses data received from the U.S. Food and Drug Administration (FDA)
and the cosmetics industry to determine expected cosmetic use. Use frequencies of individual ingredients in cosmetics are
collected from manufacturers and reported by cosmetic product category in FDA’s Voluntary Cosmetic Registration Program
(VCRP) database. Use concentration data are submitted by Industry in response to surveys of maximum reported use
concentrations, by product category, that are conducted by the Personal Care Products Council (Council). Collectively, the
use frequency and use concentration data indicate that 7 of the 10 silk protein ingredients are currently being used in cosmetic
products. According to these data, the following 3 silk protein ingredients are not being used in cosmetics:
Fibroin
MEA-Hydrolyzed Silk
Silkworm Cocoon Extract

According to 2015 VCRP data, the greatest reported use frequency is for hydrolyzed silk (675 formulations, mostly
rinse-off), followed by silk powder (177 formulations, mostly leave-on) (Table 4).32 Lower use frequencies are being
reported for the remaining silk ingredients. The results of a concentration-of-use survey conducted in 2014 indicated that silk
powder had the highest maximum concentration of use; it was used at concentrations up to 1.4% in leave-on products (face
powders) (Table 4).33 In some cases, reported uses appear in the VCRP database, but concentrations-of-use data were not
provided. For example, hydrolyzed sericin is reported as used in 4 cosmetic formulations, but use concentration data were
not submitted.

Cosmetic products containing silk proteins may be applied to the skin and hair or, incidentally, may come in contact
with the eyes and mucous membranes. Products containing these ingredients may be applied as frequently as several times
per day and may come in contact with the skin or hair for variable periods following application. Daily or occasional use
may extend over many years.

Hydrolyzed silk and silk extract are used in hairspray at maximum concentrations up to 0.024% and 0.0036%,
respectively. Silk powder is also used in hairspray (maximum concentration 0.02%). Hydrolyzed fibroin and silk powder are
used in perfume at maximum concentrations up to 0.000047% and 0.1%, respectively. Maximum use concentrations for the
following ingredients in face powders are reported: sericin (0.00047%), silk (0.1%-0.2%), and silk powder (0.1%-1.4%). In
practice, 95% to 99% of the droplets/particles released from cosmetic sprays have aerodynamic equivalent diameters >10 µm,
with propellant sprays yielding a greater fraction of droplets/particles below 10 µm, compared with pump sprays.34,35,36,37
Therefore, most droplets/particles incidentally inhaled from cosmetic sprays would be deposited in the nasopharyngeal and
bronchial regions and would not be respirable (i.e., they would not enter the lungs) to any appreciable amount.34,35

The silk proteins reviewed in this safety assessment do not appear on the list of ingredients prohibited from use in
cosmetic products marketed within the European Union (Annex II of the Council of the European Communities Council
Directive ), or on the list of ingredients with use restrictions in products marketed within the European Union (Annex III).38

Noncosmetic

Fibroin

Silk fibers made from fibroin have many uses in textiles (medical and industrial applications) mainly because of the
unique properties of fibroin, such as water absorbency, dying affinity, thermo-tolerance, luster and insulation properties.
Fibroin is also a raw material for producing precious fabrics, parachutes, tire lining materials, artificial blood vessels and
surgical sutures.10

Natural, nonabsorbable silk surgical suture containing the organic protein fibroin is an FDA-approved medical
device.39

TOXICOKINETICS

Toxicokinetics studies of the silk proteins reviewed in this safety assessment were not found in the published
literature, and unpublished data were not submitted.
 TOXICOLOGY

Single Dose (Acute) Toxicity

Oral

Hydrolyzed Silk

The acute oral toxicity of hydrolyzed silk ( m.w. ≈ 300 Da, acid and enzyme hydrolysis product) was evaluated
using rats (5 males, 5 females; strain not stated).40 A single dose of 10 g/kg was administered orally to each animal. No
signs of toxicity were observed during the 14-day observation period after dosing.

In another study, the acute oral toxicity of hydrolyzed silk protein (mw ~ 1,000 Da; produced via alkali hydrolysis)
was evaluated using albino rats (5 males, 5 females).41 A single dose of the test material (5 g/kg body weight) was
administered using an intragastric feeding needle. Signs of toxicity were not observed during the study and none of the
animals died. The LD 50 was > 5 g/kg.

The acute oral toxicity of hydrolyzed silk protein (15% to 25% in water; specific gravity = 1.10) was studied using
10 albino rats (5 males, 5 females).41 The test substance was administered orally at a dose of 5 g/kg, and dosing was followed
by a 14-day observation period. Gross necropsy was performed on all animals. Only 1 animal (male) died, and thoracic
cavity filled with fibrous tissue was noted at necropsy. Whether or not this death was due to experimental error was not
stated. Gross changes were not observed in the remaining animals. The test substance was classified as non-toxic.

Silk

Ten male Sprague-Dawley rats were dosed orally (16 g/kg) with silk.42 The form of silk administered, test
concentration, vehicle, and dosing method were not stated. Dosing was followed by a 14-day observation period. None of
the animals died, and, except for slight lethargy, there were no signs of toxicity during the observation period. The oral LD 50
was > 16 g/kg, and silk was considered nontoxic in this study.

Silk Powder

A 30% solution of silk powder in distilled water was administered orally (feeding tube) to 12 female DD-strain
mice.43 The animals received doses up to 12 g/kg body weight (dose volume = 40 ml/kg). Dosing was followed by a 7-day
observation period. Toxic signs were not observed during the study, and the test substance was classified as practically non-
toxic (LD 50 > 12 g /kg body weight).

Repeated Dose Toxicity

Dermal

Hydrolyzed Silk

In a cumulative skin irritation study involving 8 Hartley guinea pigs, a 6.5% aqueous solution of hydrolyzed silk
protein (m.w. = 650 Da) was applied to the back once daily for 35 days.44 The animals were killed and necropsied at the end
of the dosing period. Body weight gain was normal, and no abnormalities were noted at necropsy. Results relating to skin
irritation potential are included in Table 5.

Silk Powder

In a skin sensitization study, silk powder (50% in sterile water; 0.5 ml on 20 x 20 mm occlusive patch) was applied
for 6 h to the left flank of 20 young adult female guinea pigs of the Dunkin-Hartley strain.45 Ten guinea pigs served as
controls. This procedure was repeated at weekly intervals for a total of 3 weeks. Following a 2-week non-treatment period,
challenge patches were applied to the right flank for 6 h. Two animals died during the study, and necropsy results did not
indicate a test substance-related effect.
 Cytotoxicity

Sericin obtained via urea extraction was slightly toxic to mouse fibroblasts in vitro at concentrations as low as 60
µg/ml, and toxicity was substantial (i.e., severely harmful) at concentrations greater than100 µg/ml. When using other
extraction methods (heat, acid, or alkaline), sericin yielded less toxicity, as measured by the percentage of viable
fibroblasts.46

Skin Depigmentation

Sericin

Sericin was formulated as an 8% cream and applied to one side of the extremity (arm and leg) of renal patients who
normally experienced dry and itchy skin.47 A cream base was applied to the other extremity and served as the control. From
47 subjects who completed the study, the skin hydration of the patients’ extremities increased after receiving both sericin
cream and the cream base, but the changes in skin hydration were much greater on the side receiving the sericin cream than
on the side receiving the cream base. Additionally, at the end of the study, the skin pigmentation level was significantly
reduced on both the arms (p = 0.032) and legs (p = 0.021) of the sericin-treated side compared with the side treated with
cream base.

The depigmentation effect was presumed to result from tyrosinase inhibition. The degree of inhibition of tyrosinase
(the rate-limiting enzyme for melanin production) activity by sericin is known to depend upon the extraction method and silk
strain source.48 For example, colored silk cocoons, which contain flavonoids and carotenoids, exhibit higher anti-tyrosinase
activity than white-shelled cocoons.

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

Reproductive and developmental toxicity studies of the silk proteins reviewed in this safety assessment were not
found in the published literature, and unpublished data were not submitted.

GENOTOXICITY

Hydrolyzed Silk

The genotoxicity of hydrolyzed silk protein (10% aqueous) was evaluated in the Ames test using the following
Salmonella typhimurium strains, with and without metabolic activation: TA98, TA100, TA1535, TA1537, and TA1538.49
Results were negative in all bacterial strains. The positive and negative controls performed as expected in this assay.

CARCINOGENICITY

Cell Proliferation

Sericin

The effect of sericin on the rat insulinoma cell line (seeded on ASF104 culture medium [serum-free medium
containing insulin and transferrin]) was evaluated. Bovine serum albumin (BSA) served as the control protein. The RIN-5F
cell cultures were identified as follows: ASF104 (1 ml), ASF104 with 0.1% sericin, and ASF104 with 0.1% BSA. The cells
were cultured for 22 h. Viable and non-viable cell numbers were determined using the trypan blue exclusion method. The
cells in the control culture failed to proliferate. However RIN-5F cells propagated significantly in the presence of sericin (p <
0.05) or BSA (p < 0.01). Therefore, sericin and BSA were efficient inducers of RIN-5F cell proliferation.50

ANTICARCINOGENICITY

Sericin

A study was performed to evaluate the protective effect of sericin on tumor promotion in the 7,12-
dimethylbenz[a]anthracene (DMBA)-initiated and 12-О-tetradecanoylphorbol 13-acetate (TPA)-promoted mouse skin
tumorigenesis model.51 In the first experiment, sericin was applied topically to DMBA (190 nM)-initiated, female CD-1
(ICR):Crj mice (groups of 16) at doses of 2.5 mg and 5 mg twice weekly for 16 weeks. Sericin was applied 30 minutes prior
to each promotion treatment with TPA (3.2 nM). The tumor necrosis factor alpha (TNF-α, pro-inflammatory cytokine) and
4-hydroxynonenal (4-HNE) staining method was used, whereby the affinity-purified goat polyclonal TNF-α antibody and
monoclonal antibody were placed on the specimens. Sericin caused a significant reduction in both the tumor incidence and
tumor multiplicity at doses of 2.5 mg and 5 mg per application, compared to the control group that was not treated with
sericin. The expression of TNF-α protein and the level of 4-HNE in normal epidermis were significantly reduced in both
sericin treatmernt groups.

In the second experiment (groups of 5 ICR mice), sericin (5 mg) was applied topically to dorsal skin 30 minutes
prior to application of TPA. The same doses of TPA and sericin (used in first experiment) were applied twice at an interval
of 24 h. Treatment with sericin inhibited double TPA treatment-induced morphological changes that were indicative of an
inflammatory response, i.e., leukocyte infiltration, hyperplasia, and cell proliferation. Treatment with sericin also
significantly suppressed the elevation of 4-HNE levels and elevated expressions of c-fos, c-myc, and cyclooxygenase-2
(COX-2) in normal epdidermis that were induced by double application of TPA. The results of this study suggest that sericin
has a protective effect against tumor promotion in mouse skin by suppressing oxidative stress, inflammatory responses and
TNF-α.51

In another study, male CD-1 (ICR): Crj mice (2 groups of 11 and 12 respectively) were fed diets supplemented with
1.5% sericin (3 g/kg/day) or 3% sericin (6 g/kg/day) for 5 weeks.52 The animals also received weekly injections of 1,2-
dimethylhydrazine (DMH) during the initial 3 weeks of the study. The feeding of sericin in the diet resulted in a dose-
dependent decrease in the development of colonic aberrant crypt foci. In a second experiment, mice were fed a diet
supplemented with 3% sericin for 115 days. The animals were also injected with DMH weekly during the initial 10 weeks.
Both the incidence and number of colon tumors were suppressed by sericin consumption.

A study was performed to assess the protective effect of sericin on ultraviolet B light (UVB)-induced acute damage
and tumor promotion in HR-1 hairless mouse skin.53 Three groups of 10 mice were treated dermally with sericin, BSA, and
vehicle (ethanol), respectively, in the first experiment. One group of mice was treated with 180 mJ/cm2 UVB light once daily
for 7 days, after which red sunburn lesions of the skin were observed. Both the area and the intensity of the redness of these
lesions were reduced by the topical application of 5 mg sericin immediately after UVB treatment. The differences (area and
intensity of the redness) between the vehicle and sericin groups were statistically significant (p < 0.01). This was not true
when the group treated with BSA (5 mg), rather than sericin, was compared to the vehicle control. The results of
immunohistochemical analyses indicated that the application of sericin suppressed UVB-induced elevations in 4-
hydroxynonenal (4-HNE), expression of cyclooxygenase-2 (COX-2) protein, and proliferating cell nuclear antigen (PCNA)-
labeling index in the UVB-exposed epidermis.

Three groups of 15 mice of the same strain were treated with sericin, BSA, and vehicle (ethanol), respectively, in the
second experiment. One group of mice was treated (dermal application) with 200 nmol DMBA, followed by a 1-week non-
treatment period. DMBA-treated skin was then irradiated with 180 mJ/ cm2 of UVB twice weekly, and each irradiation was
followed by topical treatment with sericin (5 mg). Another group of mice was treated similarly with BSA (5 mg), rather than
sericin. Treatments (UVB dosing, followed by topical treatment) were repeated for 22 weeks. A statistically significant
reduction in both tumor incidence and multiplicity was noted at a dose of 5 mg, indicative of a suppressive effect of sericin.
When compared to all of the animals in the vehicle and BSA groups having skin tumors 22 weeks after the topical
application of DMBA, only 6% of the DMBA-exposed mice in the sericin-treated group exhibited skin tumors, indicating
94% (p < 0.001) reduction in tumor incidence. Similarly, when the tumor data were evaluated for tumor multiplicity (i.e., the
number of tumors per mouse), from the first tumor appearance to the termination of the experiment, sericin produced
statistically significant (p < 0.05) protection against UVB-induced tumor promotion in DMBA-exposed mouse skin. The
results of this study (including the first and second experiments) suggest that sericin possesses a photoprotective effect
against UVB-induced acute damage and tumor promotion by reducing oxidative stress, COX-2, and cell proliferation in
mouse skin.53

IRRITATION AND SENSITIZATION

Skin Irritation and Sensitization

Skin irritation and sensitization studies on silk protein ingredients are summarized in Table 5. These ingredients are,
at most, mild skin irritants and lack skin sensitization potential. In the only available human skin irritation/sensitization tests
in which test concentrations were reported, 20% aqueous hydrolyzed silk was a non-irritant and a non-sensitizer and 6.5%
aqueous hydrolyzed silk was a non-irritant (skin irritation only evaluated).
 Allergenicity

Human

Silk

The relationship between silk sensitization and asthma incidence was evaluated in 871 children living in China.54
Skin testing was performed using a slightly modified version of the semiquantitative puncture method. The results of
multivariate analyses of asthma incidence and skin test reactivity to aeroallergens were presented. Individual skin test results
were not provided. Children who were sensitized to silk had 2.6 times higher odds of having asthma than did nonreactors,
after adjustment for age, gender, familial correlations, and skin test reactivity to other aeroallergens using generalized
estimating equations. This association between sensitization to silk and asthma yielded lower statistical p values when the
eosinophil counts of the participants were included as either a categorical variable or a linear term in the multivariate model.

Sixty-four children (< 15 years old; males and females) with silk-induced asthma in China were studied.55 The
diagnosis was based on a history of wheezing, positive skin tests to silk, positive nasal or conjunctival provocation tests, or
serum IgE-silk waste (serum antibodies against silk waste [severely broken silk threads, used only as filling for bed quilts or
clothes and mattresses]). The average age of asthma onset was 4 years 2 months. Conjunctival provocation tests were
performed on 80% of the cases. The first symptom was observed an average of 10 months after initial exposure to silk.
Asthma was accompanied by allergic rhinitis in 61% of the patients, and was accompanied by conjunctivitis in 14% of the
cases. In most cases, asthma occurred during the winter, due to the seasonal use of bed quilts or clothes filled with silk. The
average mean wheal diameter elicited by silk in prick tests was greater than the diameters measured from 2 histamine
equivalent prick tests per silk-sensitive subject.

In relation to the preceding study, it should be noted that allergenic proteins have been extracted from one silk batch
that was imported to be used as filling material for bed mattresses and rugs.56 IgE and IgG antibodies to the extracted silk
proteins were measured by radioallergosorbent (RAST) in sera of 9 silk-sensitive subjects and in sera of healthy control
subjects. IgE and IgG antibodies to the individual silk polypeptides (40 kilodaltons (kd), 45 kd, and 70 kd) were detected
using the immunoblot technique; the 6 kd silk polypeptide bound only IgG. The sera of silk-sensitive subjects contained high
titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Low IgG antibody titers to a limited number
of these polypeptides were detected in sera of control subjects. In another study involving 10 patients with a positive skin
prick test to silkworm crude extract, arginine kinase (42-kda protein) was identified as a major allergen in this crude extract.57

Phototoxicity

Hydrolyzed Silk

The phototoxicity of 6.5% aqueous hydrolyzed silk protein (mw = 650 Da; produced by alkaline and enzyme
hydrolysis) was evaluated using groups of 6 Hartley guinea pigs.58 The 3 groups were identified as test, positive control, and
negative control groups. 8-Methoxypsoralen (1%) served as the positive control. The negative control was not stated. The
test material was applied topically (dose = 0.05 ml/2 x 2 cm) to 2 sites on dorsal skin. One site was irradiated once with a
FL-40S lamp and BLP lamp (wavelength range not stated), and the other site was covered. The sites were examined
macroscopically at 24 h, 48 h, and 72 h. Phototoxicity was evaluated based on the difference in severity of skin reactions
between the irradiated and non-irradiated sites. Hydrolyzed silk protein was classified as non-phototoxic. Positive
responses were observed in all of the guinea pigs treated with 8-methoxypsoralen + light.

Silk Powder

Silk powder (0.1 g) was applied to the back (2 sites [2 areas per site]) of each of 6 female guinea pigs of the Hartley
strain.43 The test site (cm2 area not stated) was covered with a patch plaster for 4 h, after which the sites were irradiated with
UV light (minimal erythema dose, 15-minute exposure) from a 20-watt lamp. The test sites were evaluated for erythema and
eschar formation after 24 h and 48 h. Positive reactions were not observed in this study.

Photoallergenicity
Hydrolyzed Silk

The photoallergenicity of 6.5% aqueous hydrolyzed silk protein (mw = 650 Da; produced by alkaline and enzyme
hydrolysis) was evaluated using groups of 6 Hartley guinea pigs.59 The 3 groups were identified as test, positive control, and
negative control groups; the positive control was 3,5,4'-tribromosalicylanilide (2% in 85% dimethylsulfoxide),but the
negative control was not stated. The test material was applied transdermally (0.05 ml /2 x 2 cm), with or without UV
irradiation, 5 times per week (2 h per day) for a total of 10 applications. Applications were made on both sides of the dorsal
area, symmetrically. One side was irradiated for 2 h, and the other side was covered. The photochallenge phase was
initiated after a 2-week non-treatment period. The test material was applied to 2 sites. One side was irradiated for 2 h, and
the other side was covered. The application site was examined macroscopically after the challenge and 24 h, 48 h, and 72 h
later. No effects were observed in negative controls or in guinea pigs treated with the test material (with or without exposure
to light). Hydrolyzed silk protein was considered non-photosensitizing in this study. The expected results were achieved
with the positive control.

Case Reports

According to one case report, recurrent granulomas with remarkable infiltration of eosinophils may have resulted
from an IgE-mediated hypersensitivity reaction to silk fibroin, a component of the braided silk suture used. 60 In this report, a
lateral skin flap technique had been performed to correct tracheostomal stenosis, using silk sutures, after a total
laryngectomy.

Adverse reactions to virgin silk sutures in 12 cataract surgery patients have also been reported.61 Nodular
episcleritis, peripheral corneal ulceration, and wound necrosis with dehiscence were observed, sometimes resulting in
endophthalmus or epithelial down-growth. Conjunctival and scleral histopathologic studies in 4 eyes showed acute and
chronic inflammation with multinucleated giant cells. Type I allergic responses and up-regulated levels of specific IgE were
reported to occur in patients after repeated surgical procedures.60,62

A female patient with a history of severe atopic dermatitis and various allergies, and a family history of atopic
eczema and asthma, presented with exacerbation of eczema on her hands and wrists and urticarial papules on the flexor
aspects of both forearms.63 The same types of lesions had been observed on both arms after wearing the same silk shirt, but
quickly disappeared after the shirt was taken off. RAST tests yielded positive results for silk waste (k73) of 2.08 universal
arbitrary units (Ua)/ml and positive results for silk (k74) of 3.62 Ua/ml. The total IgE count was > 5000 kilo units (kU)/l.
These results confirmed the diagnosis of immunological contact urticarial caused by silk.

Ocular Irritation

Animal

Hydrolyzed Silk

The ocular irritation potential of hydrolyzed silk protein (15% to 25% in water) was evaluated in the Draize test
using 6 New Zealand white rabbits.64 The test substance (0.1 ml) was instilled into one eye of each animal, and eyes were
not rinsed. Contralateral eyes served as controls. Observations for ocular reactions were made up to 72 h post-instillation.
The test substance was practically non-irritating to the eyes of rabbits.

The ocular irritation potential of 6.5% aqueous hydrolyzed silk (mw ~ 300 Da) was studied using 6 New Zealand
white rabbits.65 The test material (0.1 ml) was instilled into the right eye of each animal, and the left eye served as the
untreated control. Reactions were scored at 24 h, 48 h, and 72 h post-instillation. Slight conjunctival rednesss, the only
reaction reported, was observed in one rabbit. It was concluded that it is not likely that hydrolyzed silk would be classified as
an ocular irritant, according to the definitions of the U.S. Federal Hazardous Substances Act. The ocular irritation potential
of a higher molecular weight hydrolyzed silk (mw = 650 Da; test concentration not stated) was evaluated in New Zealand
white rabbits according to the same test procedure, and the results were negative.66

Hydrolyzed silk (mw ~ 1,000 Da; produced by alkali hydrolysis) was placed (0.1 ml) in the right eye of each of 6
New Zealand white rabbits.41 Observations for any signs of corneal opacity, iritis, or conjunctivitis were made at 24 h, 48 h,
and 72 h post-instillation. The authors concluded that hydrolyzed silk was practically non-irritating to the eyes of rabbits.

In a cumulative ocular irritation test, 6.5% aqueous hydrolyzed silk protein (mw = 650 Da; 0.1 ml; produced by
acid, alkaline, and enzyme hydrolysis), was instilled into the eyes of 6 New Zealand white rabbits three times per day for 4
days continuously.67 Conjunctival redness was observed in 5 of 6 animals at 3 or 4 days. Reactions in the cornea or iris were
not observed. The authors concluded that hydrolyzed silk protein was practically non-irritating to the eyes of rabbits.
Silk

Silk (0.1g) was instilled into one eye of each of 9 adult albino rabbits.42 The eyes of 3 rabbits were rinsed
immediately after instillation. Untreated eyes served as controls. The eyes were examined at 24 h, 48 h, and 72 h post-
instillation. Transient conjunctival redness (unrinsed eyes) was observed in 5 of 6 rabbits. However, no effects on the cornea
or iris were observed. Ocular irritation was not observed in the 3 rabbits subjected to ocular rinsing. Silk was classified as a
non-irritant in this study.

Silk Powder

The ocular irritation potential of a silk powder solution (10% in saline) and the supernatant fluid from this solution
(filtered after 24 h) was evaluated using a total of 6 white rabbits.43 Four rabbits received the 10% in saline solution and 6
rabbits received the supernatant. Either test substance (0.1 ml) was instilled into the right eye, and eyes were rinsed.
Untreated left eyes served as controls. The eyes were examined for reactions for up to 168 h post-instillation. The silk
powder solution (10% in saline) was classified as slightly positive. The supernatant from this solution was not an ocular
irritant.

In Vitro

Hydrolyzed Silk

The ocular irritation potential of hydrolyzed silk protein (mw = 300 Da; 2% active solution
n) was evaluated in the in vitro hen’s egg test on the chorioallantoic membrane (HET-CAM).16,68 The material (0.3 ml) was
tested on the choroallantoic membrane of fertilized Leghorn hens’ eggs that had been incubated for 10 days. Results were
negative (score = 0.3).

Hydrolyzed silk protein (mw = 300 Da; 10% active solution) was tested for ocular irritation potential in the in vitro
red blood cell aggregation test (RBCA), which evaluates effects on the cytoplasmic membrane.16,69 A total irritation
classification was obtained by determining the hemolysis/denaturation (L/D) ratio. A substance with an L/D of > 100 was
classified as a non-irritating. Hydrolyzed silk caused neither hemolysis nor denaturation, and was classified as non-irritating.

The Irritection® assay was used to evaluate the ocular irritation potential of hydrolyzed silk.70 The test material was
applied to the Irritection® system at dose volumes of 25 µl, 50 µl, 75 µl, 100 µl, and 125 µl. The samples remained at room
temperature for 24 h and were then analyzed by spectrophotometry. Over the range of dose volumes tested, ocular Irritection
scores for hydrolyzed silk ranged from 2.5 to 3.5. Scores in this range corresponded to a classification of minimally
irritating.

The ocular irritation potential of hydrolyzed silk was studied using the EpiOcularTM model assay.71 The test material
was applied to a reconstructed human corneal epithelial model for 30 minutes, and cell viability was measured by
dehydrogenase, present in the cell mitochondria, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), into blue, MTT formazan salt. The irritation potential of the test material is dictated by the reduction in tissue
viability of exposed tissues, compared to the negative control (sterile deionized water). Methyl acetate served as the positive
control. Hydrolyzed silk was classified as a non-irritant. The negative and positive controls were non-irritating and
irritating, respectively.

OTHER EFFECTS

Immunological Responses

Sericin and Fibroin

In Vitro

Soluble sericin proteins (2 µg of sericin per well tissue culture plate) extracted from native silk fibers did not induce
significant macrophage activation.72 Macrophages exposed in vitro to the silk preparations failed to respond with consistently
elevated levels of TNF in either short- or long-term cultures. However, the suspension of the crystalline particles prepared by
enzymatic digestion of silk fibroin was the only silk preparation that yielded significant TNF release, which was probably a
non-specific response to insoluble physical particulates, rather than a specific, chemically-induced response to silk. Whether
or not the statistical significance of this finding was determined was not stated. However, it was noted that the average TNF
release (corrected for volume and expressed as total release from specified cell count) and standard error of the mean were
determined.

A study was designed to investigate the inflammatory mediators induced by sericin.73 Sericin was analyzed in an in
vitro macrophage (mouse alveolar macrophage cell line) and monocyte (mouse monocyte cell line) assay to assess cytokine
activation. These cell lines were used for monitoring levels of interleukin (IL-1β) and tumor necrosis factor (TNF-α)
generated after activation by sericn at concentrations of 0.2 to 1.0 mg/mL. Inflammatory mediators activated by sericin were
also investigated in vivo, using a rat wound-healing model. At the concentrations tested, silk sericin increased the amounts of
inflammatory mediators and proinflammatory cytokines, TNF-α and IL-1β (involved in the modulation of skin growth, repair
and scarring during inflammation). However, the maximum levels of TNF-α and IL-1β released from monocytes and
macrophage cells after silk sericin exposure were 500 and 350 pg/ml, respectively. It was noted that these levels of cytokines
would not be sufficient to cause an inflammatory response or prevent cellular proliferation.

The suppression of inflammation by sericin has been reported.74 Sericin solution (0.004 to 0.080 mg/ml) applied
topically to the top of the hind paw of rats prior to a carrageenan subcutaneous injection under the plantar surface of the hind
paw exhibited anti-inflammatory activity, similar to the effect of indomethacin (a non-steroidal anti-inflammatory drug used
as a control). The amount of mast cells in rat tissue treated with sericin or indomethacin was much lower compared to the
amount of cells found in tissue treated with water (control). Further investigation indicated that sericin did not cause a
hypersensitivity reaction. On the contrary, it inhibited cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase
(iNOS) production (monitored by total RNA and real-time polymerase chain reaction (RT-PCR)) in fibroblast cell culture,
resulting in lowering the inflammation of the carrageenan induction.

Wound Healing

Non-human

Sericin

The effect of a sericin cream on wound healing was evaluated using 18 male Sprague-Dawley rats (8 weeks old).75
The composition of the cream was described as follows: 8% sericin, white petrolatum, mineral oil, lanolin, glycerin,
bisabolol, propylparaben, and methylparaben. Except for sericin, the concentration of each cream component was not stated.
The cream was applied topically to full-thickness skin wounds on the dorsum of each animal, and wound surfaces were
observed for 15 days post-application. Cream base without sericin served as the control. Histological examination of
wounds after 15 days of treatment with 8% sericin cream revealed complete healing, no ulceration, and an increase in
collagen, as compared to treatment with the control cream. Wounds treated with the control cream had some ulceration and
acute inflammatory exudative materials.

In a similar study involving 45 Sprague-Dawley rats (8 weeks old), 8% sericin cream was applied to full-thickness
wounds on the dorsum of each animal. Cream base without sericin served as the control. Excised rat tissue was prepared for
cytokine determination. IL-1β and TNF-α are proinflammatory cytokines that are involved in a variety of immunological
functions. Wounds treated with sericin cream did not yield significantly high levels of IL-1β and TNF-α on day 7, which
suggests that the cream did not induce an inflammatory or immunological response.76

In Vitro

Sericin

Human skin fibroblasts were incubated with sericin in vitro for 72 h.77 The cell count in treated cultures after 72 h
was enhanced to 250% of the untreated (i.e., no-sericin) control cultures. In another study, replacing the culture medium with
sericin solution in the mouse L929 fibroblastic cell line in culture increased the percentage of cell proliferation significantly,
especially at a high sericin concentration (1.0 mg/ml).73 The amount of NF-1α and IL-1β released from alveolar macrophage
NR8383 and mouse J774.2 monocyte cell lines after the addition of sericin (0.2 - 1.0 mg/ml) to the culture media was
negligible, indicating that sericin did not cause severe damage to the cells.

SUMMARY

The safety of the following 10 silk protein ingredients in cosmetics is reviewed in this safety assessment: fibroin,
hydrolyzed fibroin, hydrolyzed sericin, hydrolyzed silk, MEA-hydrolyzed silk, sericin, silk, silk extract, silk powder, and
silkworm cocoon extract. These ingredients are reported to function as skin and hair conditioning agents and bulking agents
in cosmetic products. Frequency of use data from the FDA’s VCRP and the results of an industry survey indicate that 7 of
the 10 silk protein ingredients are being used in cosmetic products. Silk powder has the highest reported maximum
concentration of use; it is used at concentrations up to 1.4% in leave-on products (face powders).

The silkworm, Bombyx mori, produces silk proteins during the final stage of larval development, and two silk
proteins, fibroin and sericin, have been distinguished as major components of silk cocoons. In the process of manufacturing
silk, fibroin is separated from sericin by a degumming process. There are several methods for removing sericin in the
degumming process of cocoons. Because the method of production of sericin involves extraction from cocoons using soaps
and detergents, detergents and alkali soaps and are typically present as impurities in sericin.

Hydrolyzed silk is prepared from the cocoon of Bombyx mori. Hydrolyzed silk protein (mw = 300 Da) may be
prepared by acid, alkaline, or enzyme hydrolysis; hydrolyzed silk protein (650 Da) may be prepared by alkaline and enzyme
hydrolysis.

In acute oral toxicity studies involving rats, LD 50 values of > 10 g/kg body weight (hydrolyzed silk protein, mw ~
300 Da), > 5 g/kg (hydrolyzed silk protein, mw ~ 1,000 Da), > 16 g/kg (silk), and > 12 g/kg (30% aqueous silk powder) were
reported. Signs of toxicity were not observed.

In a repeated insult patch test (RIPT, occlusive patches) involving dermal applications of silk powder (50% in sterile
water) to 20 guinea pigs over a 3-week period, 2 animals died. However, necropsy results were not indicative of a test
substance-related effect. Similarly, no abnormalities were noted at necropsy in a cumulative skin irritation in which 6.5%
aqueous hydrolyzed silk (mw=650 Da) was applied to 8 Hartley guinea pigs once daily for 35 days.

Sericin obtained via urea extraction was toxic to mouse fibroblasts in vitro at concentrations as low as 60 µg/ml.
When compared to urea extraction, the extraction of sericin by other methods resulted in reduced toxicity to mouse
fibroblasts in vitro.

In the Ames test, results for 10% aqueous hydrolyzed silk protein were negative.

Sericin and BSA were efficient inducers of rat RIN-5F cell proliferation in a rat insulinoma cell line. The results of
another study involving CD-1 (ICR):Crj mice suggested that sericin had a protective effect against tumor promotion in mouse
skin by suppressing oxidative stress, inflammatory responses and TNF-α.

The results of a study in mice suggested that sericin possesses a photoprotective effect against UVB-induced
damage and tumor promotion by reducing oxidative stress and cell proliferation in mouse skin. In another study involving
mice, the feeding of diets supplemented with 1.5% or 3% sericin for 5 weeks resulted in a dose-dependent decrease in the
development of colonic aberrant crypt foci.

Undiluted hydrolyzed silk protein (mw ~ 300 Da; dose = 0.5 ml/2.5 cm2) caused reactions ranging from very slight
to well-defined erythema on intact and abraded skin of rabbits. However, a primary irritation index (PII) of 1.1 was reported,
and the test material was not classified as a primary skin irritant. Hydrolyzed silk protein (mw ~ 1,000 Da; dose = 0.5 ml/
2.5cm2; PII = 0.65) and hydrolyzed silk protein (mw = 650 Da; dose = 0.5/2.5 cm2; PII = 0.05) were also classified as non-
irritating to the skin of rabbits. Hydrolyzed silk protein (15% to 25% aqueous) was non-irritating to the skin of rabbits. In a
cumulative skin irritation study involving guinea pigs, hydrolyzed silk protein (mw = 650 Da) was considered non-irritating
to the skin. Hydrolyzed silk protein (mw = 650) did not induce skin sensitization in a study involving guinea pigs. Silk
powder (0.5 g) was non-irritating to abraded or intact skin of rabbits, and silk powder (0.1 g) was non-irritating and non-
sensitizing to the skin of guinea pigs.

There was no evidence of skin irritation in 6 rabbits after the application of silk (0.5 g) under a 2 cm2 patch for 24 h.
Silk powder (0.5% w/v in distilled water) did not induce sensitization in an RIPT involving 20 guinea pigs. Also, the results
of a preliminary skin irritation test at concentrations up to 10% in distilled water were negative. Similarly, silk powder (50%
in sterile water) did not induce sensitization in an RIPT involving 20 guinea pigs. Furthermore, the results of a preliminary
skin irritation test indicated that 75% silk powder was reasonably tolerated by the 5 guinea pigs tested.

Negative results were reported for hydrolyzed silk protein (mw = 300 Da) in a skin irritation study involving 20
subjects, and for hydrolyzed silk protein (mw = 650) in a study involving 24 subjects. In two RIPT’s involving 57 subjects
and 49 subjects, respectively, hydrolyzed silk protein (mw ~ 1,000 Da; dose ~ 0.2 ml/1" x 3/4" area and dose ~ 2 ml/4 cm2
area, respectrively) was not classified as a skin irritant or sensitizer. In an HRIPT involving 48 subjects, the results relating
to the skin irritation and sensitization potential of hydrolyzed silk protein (mw ~ 300 Da; dose = 20 µl/40 mm2 Finn chamber)
were negative. Silk powder (0.05 g) was non-irritating to the skin of 30 subjects.
 Results for hydrolyzed silk were negative for skin irritation potential in the Irritection® (dose volumes up to 125 µl)
and EpidermTM (aqueous solution containing 27% to 32% hydrolyzed silk) in vitro assays. The mouse local lymph node
assay yielded negative results relating to the sensitization potential of 20% aqueous hydrolyzed silk protein (mw = 300 Da).

Hydrolyzed silk protein (mw = 650 Da; dose = 0.05 ml/2 x 2 cm) was neither phototoxic nor photoallergenic to the
skin of guinea pigs. Silk powder (0.1 g) also was not phototoxic when applied to the skin of guinea pigs.

Hydrolyzed silk protein of an average molecular weight of ~ 300 Da, 650 Da, or ~ 1,000 Da did not induce ocular
irritation when instilled into the eyes of rabbits. The tests involving hydrolyzed silk protein (mw ~ 300 Da or ~ 1,000 Da)
were single-instillation tests, whereas, hydrolyzed silk protein (mw = 650 Da) was instilled 3 times per day for 4 days. In
another test, hydrolyzed silk protein (15% to 25% aqueous, single instillation) was non-irritating to the eyes of rabbits.
Hydrolyzed silk protein (mw = 300 Da; 2% active solution) was also negative for ocular irritation potential in the HeT-CAM,
Irritection®, and EpidermTM in vitro assays. Silk was also classified as a non-irritant when instilled into the eyes of rabbits,
and the results for 10% aqueous silk powder were slightly positive when instilled into the eyes of rabbits.

The suppression of inflammation by sericin was reported in a study on rats, and a hypersensitivity reaction was not
observed.

An association between sensitization to silk and asthma incidence was found in a study of 871 children. In another
study of 64 children, the average mean wheal diameter elicited by silk in prick tests was greater than 2 histamine equivalent
prick tests. Allergenic proteins have been extracted from a silk batch imported for use as filling material for bed mattresses
and rugs. IgE and IgG antibodies to the extracted silk proteins were measured in the sera of 9 silk-sensitive subjects, and
these sera were found to contain high titers of IgE and low titers of IgG antibodie to the separated silk polypeptides. In a
study involving 10 patients with a positive skin prick test to silkworm crude extract, arginine kinase was identified as a major
allergen in this crude exract.

A female patient with a history of severe atopic dermatitis and various allergies, and a family history of atopic
eczema and asthma, presented with exacerbation of eczema on her hands and wrists and urticarial papules on the flexor
aspects of both forearms.63 RAST tests yielded positive results for silk waste (k73) of 2.08 universal arbitrary units (Ua)/ml
and positive results for silk (k74) of 3.62 Ua/ml. The total IgE count was > 5000 kilo units (kU)/l. These results confirmed
the diagnosis of immunological contact urticarial caused by silk.

In a case report, recurrent granulomas with remarkable infiltration of eosinophils may have resulted from an IgE-
mediated hypersensitivity reaction to silk fibroin. Additionally, type I allergic responses and up-regulated levels of specific
IgE have been reported in patients after repeated surgical procedures that involved the use of silk sutures. In another case
report, the diagnosis of immunological contact urticarial caused by silk was confirmed.

Skin depigmentation has been observed in renal patients after application of an 8% sericin cream for treatment of
dry and itchy skin.

Histological examination of wounds in rats after 15 days of treatment with 8% sericin cream revealed complete
healing, no ulceration, and an increase in collagen, as compared to treatment with the control cream.

DISCUSSION

After reviewing the available data, concerns pertaining to skin depigmentation, allergenicity, inhalation exposure,
and the insufficiency of data on certain silk protein ingredients were discussed by the Panel.

The Panel reviewed a study in which an 8% sericin cream applied to the skin of renal patients caused
depigmentation, but agreed that a review of ingredients for drug effects (i.e., noncosmetic effects, such as depigmentation) 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 silk protein ingredients in cosmetic products. Sericin is used at a maximum concentration of
0.00047% in cosmetic products.

Studies on patients inhalationally exposed to silk waste, which is distinct from the silk protein ingredients that are
being evaluated in this safety assessment, showed an association between asthma and dermal allergies to silk in children in
China. The Panel determined that the results of these studies do not support a cause-and-effect relationship of silk protein
ingredients in this safety assessment and the development of asthma.
 Concern about pesticide residues and heavy metals that may be present in ingredients was expressed by the Panel.
They stressed that the cosmetics industry should continue to use current good manufacturing practices (cGMPs) to limit
impurities.

Some of the silk protein ingredients are used in products that could be incidentally inhaled. For example, hydrolyzed
silk and silk extract are used in hairspray at maximum concentrations up to 0.024% and 0.0036%, respectively, and the
highest maximum use concentration for ingredient use in face powders is being reported for silk powder (1.4%). However,
the Panel did not express concern over the use of these ingredients in formulations that might be inhaled. The Panel also
noted that in aerosol products, 95% – 99% of droplets/particles would not be respirable to any appreciable amount.
Furthermore, droplets/particles deposited in the nasopharyngeal or bronchial regions of the respiratory tract present no
toxicological concerns based on the chemical and biological properties of these ingredients and their low use concentrations.
Coupled with the small actual exposure in the breathing zone and the concentrations at which the ingredients are used, the
available information indicates that incidental inhalation would not be a significant route of exposure that might lead to local
respiratory or systemic effects. A detailed discussion and summary of the Panel’s approach to evaluating incidental inhala-
tion exposures to ingredients in cosmetic products is available at http://www.cir-safety.org/cir-findings.

The Panel determined that the available data are insufficient for evaluating the safety of MEA-hydrolyzed silk and
silkworm cocoon extract in cosmetic products and that the following data on these ingredients are needed:

• Method of manufacture and impurities

• Concentration of use
• 28-day dermal toxicity study; if absorbed, genotoxicity and reproductive and developmental toxicity data may be
needed
• Skin irritation and sensitization data

CONCLUSION

The CIR Expert Panel concluded that the following 8 ingredients are safe in the present practices of use and
concentration in cosmetics, as described in this safety assessment.

fibroin* sericin
hydrolyzed fibroin silk
hydrolyzed sericin silk extract
hydrolyzed silk silk powder

The Panel also concluded that the available data are insufficient for determining the safety of two silk protein ingredients in
cosmetic products, MEA-hydrolyzed silk* and silkworm cocoon extract*.

*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.
 Table 1. Definitions and functions of the ingredients in this safety assessment.1
Ingredient/CAS No. Definition Function
Fibroin Fibroin is a protein filament produced by the silkworm, Bombyx mori which together Bulking
9007-76-5 with Sericin composes Silk. Agents

Hydrolyzed Fibroin Hydrolyzed Fibroin is the hydrolysate of Fibroin derived by acid, enzyme or other Hair
method of hydrolysis. Conditioning
Agents; Skin-
Conditioning
Agents -
Miscellaneous
Hydrolyzed Sericin Hydrolyzed Sericin is the hydrolysate of Sericin derived by acid, enzyme or other Hair
870616-36-7 method of hydrolysis. Conditioning
73049-73-7 Agents; Skin-
Conditioning
Agents -
Miscellaneous
Hydrolyzed Silk Hydrolyzed Silk is the hydrolysate of silk protein derived by acid, enzyme or other Hair
73049-73-7 method of hydrolysis. Conditioning
96690-41-4 Agents; Skin-
Conditioning
Agents -
Miscellaneous
MEA-Hydrolyzed Silk MEA-Hydrolyzed Silk is the monoethanolamine salt of Hydrolyzed Silk (q.v.). Hair
Conditioning
Agents; Skin-
Conditioning
Agents -
Miscellaneous
Sericin Sericin is a protein isolated from the silk produced by the silk worm, Bombyx mori. Hair
60650-88-6 Conditioning
60650-89-7 Agents; Skin-
Conditioning
Agents -
Miscellaneous
Silk Silk is the fibrous protein obtained from cocoons of the silk worm. Bulking
Agents
Silk Extract Silk Extract is the extract of silk fiber. Skin-
91079-16-2 Conditioning
Agents -
Miscellaneous
Silk Powder Silk Powder is finely pulverized silk. Bulking
9009-99-8 Agents; Skin-
Conditioning
Agents -
Miscellaneous;
Slip Modifiers
Silkworm Cocoon Extract Silkworm Cocoon Extract is the extract of the cocoon of the silkworm, Bombyx mori. Skin-
91079-16-2 Conditioning
Agents -
Humectant
 Table 2. Properties of Silk Proteins
Property Value Background Information
Sericin

Form Powder; amorphous structure.10 Transforms into a β-sheet structure in presence of water.10

Easily dissolves in water at 50°C to 60°C; returns to gel form on cooling.2

Gelation is rapid at 108°C and pH ≈ 6 to 7.10

Molecular Weight 10 to > 400 kDa.10 Depending on extraction methods, temperature, pH, and processing time.10
35 to 150 kDa.10 Heat and acid extraction.10
10
15 to 75 kDa. Alkaline solution extraction.10
10 to > 225 kDa.10 Urea extraction.10
< 20 kDa.10 Recovered during early stages of raw silk production.10

> 20 kDa.10 Obtained from later stages of raw silk production.10

Decreases when molecules are transformed from random coil into the ß-sheet
Solubility Highly soluble in water.31 structure.2 IsoelectricPoint ≈ 4, because there are more acidic than basic
amino acids in sericin.28

Fibroin
Form Pale yellow mass.12
Molecular Weight 300 to 420 kDa.78

Soluble in concentrated alkalies,

concentrated mineral acids, and in
Solubility ammoniacal nickel oxide solution.
Insoluble in water, alcohol, ether,
and dilute alkalies.12

Hydrolyzed Fibroin
Form Yellow solution.79 Acid hydrolysis usually causes fibroin solution to turn yellow, and chemical
changes in amino acids such as tryptophan and tyrosine are generally
considered as the main reason for yellowing. Trytophan and tyrosine become
yellow upon hydrolysis, and the same is true for serine and glycine. Serine
and threonine beak down easily during hydrolysis, and other amino acids in
fibroin decompose in the following order: tyrosine, methionine, cysteine,
phenylalanine, and tryptophan.79

Hydrolyzed Silk
Form
Amber liquid.24
Odor Characteristic.24
Molecular Weight < 10,000 Da.25; 2,000-4,000 Da.26;
300 Da and 650 Da16,17
Solubility
Soluble in water.25
Density ( at room
temperature) 1.05 to 1.11 g/ml.24

Silk

Appearance White to slightly gray powder.14

Particle Size 5 to 15 µm.14
 Table 3. Composition Data on Hydrolyzed Silk.22,23
Silk Hydrolysate
(g/100 g protein)
Cysteic Acid 0
Hydroxpyroline 0
Aspartic Acid 7.4
Threonine 3.9
Serine 17.4
Glutamic Acid 3.6
Proline 0.9
Glycine 20.6
Alanine 21
Cystine 0
Valine 3.7
Methionine 0.4
Isoleucine 0.8
Leucine 1.2
Tyrosine 12.2
Phenylalanine 2
Lysine 1.6
Histidine 0.9
Arginine 2.4
Tryptophan NR
Lysinoalanine NR
NR = Not Reported
 Table 4. Frequency and Concentration of Use According to Duration and Type of Exposure.32,33
Hydrolyzed Silk Hydrolyzed Fibroin Sericin
# of # of # of
Uses Conc. (%) Uses Conc. (%) Uses Conc. (%)
Totals/Conc. Range 675 0.0000007-0.5 1 0.001-0.045 44 0.00047
Duration of Use
Leave-On 348 0.0000007-0.23 1 0.001 30 0.00047
Rinse off 322 0.0000007-0.5 NR 0.045 14 0.00047
Diluted for (bath) Use 5 0.0001-0.0003 NR NR NR NR
Exposure Type
Eye Area 36 0.0000007-0.23 NR NR 10 0.00047
Incidental Ingestion 3 NR NR NR NR NR
Incidental Inhalation- Sprays NR 0.0005-0.024 1*** NR 1 0.000047
Incidental Inhalation- Powders NR 0.00023-0.11** 1*** 0.001** 1 0.000047
Dermal Contact 213 0.0000007-0.11 1 NR 12 0.000047
Deodorant (underarm) NR NR NR NR NR NR
Hair - Non-Coloring 189 0.00002-0.5 NR NR NR 0.000047
Hair-Coloring 6 0.00022-0.008 NR 0.045 12 NR
Nail 1 0.01-0.03 NR NR NR NR
Mucous Membrane 126 0.0001-0.035 NR NR 1 NR
Baby Products 4 0.0003 NR NR NR NR
Silk Silk Extract Silk Powder
# of # of # of
Uses Conc. (%) Uses Conc. (%) Uses Conc. (%)
Totals/Conc. Range 27 0.000005-0.2 13 0.0013-0.015 177 0.0001-1.4
Duration of Use
Leave-On 23 0.000005-0.2 7 0.0036-0.015 163 0.0001-1.4
Rinse off 4 NR 6 0.0013-0.015 14 0.0001-0.1
Diluted for (bath) Use NR NR NR NR NR NR
Exposure Type
Eye Area 1 0.000005 1 NR 33 0.05-1.1
Incidental Ingestion NR NR NR NR 16 0.05-1.1
Incidental Inhalation- Sprays NR NR 3* 0.0036 1 0.005-0.1
Incidental Inhalation- Powders 4 0.1-0.2 NR 0.1-0.2 60 0.1-1.4
Dermal Contact 17 0.000005-0.2 1 0.0013 67 0.0001-1.4
Deodorant (underarm) NR NR NR NR NR 0.005
Hair - Non-Coloring NR NR 7 0.0036-0.015 6 0.01-0.048
Hair-Coloring NR NR NR NR NR NR
Nail 5 0.001-0.05 NR NR 7 0.0001-0.025
Mucous Membrane 3 NR NR NR 19 0.01-1.1
Baby Products NR NR NR NR NR NR
 Table 4. Current Frequency and Concentration of Use According to Duration and Type of Exposure.32,33
Hydrolyzed Sericin
# of
Uses Conc. (%)
Totals/Conc. Range 4 NR
Duration of Use
Leave-On 4 NR
Rinse off NR NR
Diluted for (bath) Use NR NR
Exposure Type
Eye Area 1 NR
Incidental Ingestion NR NR
Incidental Inhalation- Sprays 1* NR
Incidental Inhalation- Powders NR NR
Dermal Contact 1 NR
Deodorant (underarm) NR NR
Hair - Non-Coloring 2 NR
Hair-Coloring NR NR
Nail NR NR
Mucous Membrane NR NR
Baby Products NR NR
NR = Not Reported; Totals = Rinse-off + Leave-on + Diluted for (Bath) Use Product Uses.
*It is possible that these products may be sprays, but it is not specified whether the reported uses are sprays.
**It is possible that these products may be powders, but it is not specified whether the reported uses are powders.
***Not specified whether a powder or spray, so this information is captured for both categories of incidental inhalation.
Note: Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure
type uses may not equal the sum total uses.
 Table 5. Skin Irritation/Sensitization Potential of Silk Ingredients
Skin Irritation and Sensitization - Non-Human

Hydrolyzed Silk

6 female New Zealand albino rabbits. Undiluted test material applied (0.5 ml) for 24 h to abraded or intact skin using 2.5 cm2 occlusive patch.
Reactions (scored 1 h after patch removal) ranged from very slight to well-defined erythema at both intact and abraded test sites. Primary irritation score
(6 rabbits) = 1.1. According to Draize system, combined averages (primary irritation scores) of 2 or less classified as mildly irritating. Mild skin irritant.80

6 New Zealand white rabbits. 15% to 25% aqueous solution (0.5 ml) applied for 24 h to abraded and intact sites on trunk (2.5 cm2 area per site) using
occlusive patch. The test sites examined at 24 h and 72 h post-application. Non-irritant (PII = 0.65).41

6 New Zealand white rabbits. 6.5% aqueous solution applied to back (2.5 x 2.5 cm area) according to preceding test procedure. Very slight erythema in
one rabbit. Non-irritant (PII = 0.05).81

8 Hartley guinea pigs. 6.5% aqueous solution applied to back once daily for 35 days. After 31 days, the only reaction observed was very slight erythema
in 3 animals. Non-irritant.44
Groups of 6 Hartley guinea pigs. 6.5% aqueous solution. Maximization test. Subcutaneous injection and dermal application during induction. Dose per
cm2 not stated. 24-h occlusive challenge patch (0.2 ml test material) applied to dorsal skin on day 14. Reactions scored at 24 h and 48 h after challenge
patch removal. 1 of 6 animals had moderate erythema and 2 of 6 animals had slight erythema at 24 h after patch removal. Two of 6 animals had slight
erythema at 48 h after patch removal. Non-sensitizer. Positive control (0.1% 4-dinitrochlorobenzene) induced sensitization.82

Mouse local lymph node assay (OECD Guideline No. 429). 20% aqueous hydrolyzed silk (mw = 300 Da) was non-sensitizer.16,83

Silk
6 adult new Zealand albino rabbits. Test material (0.5 g in solvent [unnamed]) applied to intact area and abraded area on the back for 24 h; each site
covered with 2 cm2 occlusive patch. Reactions scored at 24 h and 72 h. No evidence of erythema, eschar, or edema at abraded or intact sites. Silk classified
as non-irritant.42
Silk Powder

6 female albino rabbits. Test material (0.5 g, under an occlusive patch) was applied for 4 h to abraded and intact sites (1 inch2 area) on the back. Sites
examined at 24 h. No skin irritation reactions at intact or abraded skin sites.43

Female guinea pigs (number of animals and strain not stated). Test material (0.1 g) applied to back (cm2 area not stated) 5 times per week for 13
weeks. No irritation or sensitization reactions.43

20 Hartley albino guinea pigs (10 males, 10 females). Test material (5% w/v in distilled water; 0.5 ml on occlusive patch [Hill Top Chamber®]) applied
for 6 h to left shoulder once per week for a total of three 6-h applications. Dose per cm2 not stated. After 2-week non-treatment period, challenge patch
applied for 24 h to new site. After patch removal, depilatory applied to challenge site for 30 minutes. Reactions scored at ~ 2 h after depilation. Ten
guinea pigs (controls) tested with distilled water. Non-irritant and non-sensitizer (test and controls).84

20 female Dunkin-Hartley guinea pigs. Test material (50% in sterile water; 0.5 ml on 20 x 20 mm occlusive patch) applied repeatedly for 6 h to left
flank (induction). Reactions were scored at 24 h after patch removal. This procedure repeated at weekly intervals (Days 8 to 9 and 15 to 16 of study). At
dfay 29, challenge patch applied to right flank for 6 h. Reactions scored 24 h and 48 h after patch removal. Sterile water applied to 10 control guinea pigs.
Non-sensitizer (test and control animals). Silk powder (75%) reasonably tolerated in preliminary skin irritation test involving 5 guinea pigs.45

Skin Irritation and Sensitization - Human

Hydrolyzed Silk
20 subjects (2 men, 18 women). 20% aqueous hydrolyzed silk (~ 3 mg on occlusive patch [Finn chamber]) applied to back for 48 h. Dose per cm2 not
stated. At 30 minutes after patch removal, mild erythema observed in 3 subjects. Mild skin irritation in only 1 of the 3 subjects at 24 h post-removal. No
skin irritation in remaining 17 subjects.16,85
24 subjects (10 men, 14 women). 6.5% aqueous solution (0.2 ml on occlusive patch) applied to upper back for 24 h. Dose per cm2 not stated. Classified as
a non-irritant.86
57 male and female subjects. HRIPT. During induction, hydrolyzed silk (~ 0.2 ml; concentration not stated) applied to upper back repeatedly using a 1"
x 3/4" semi-occlusive patch. Challenge patch applied to original site and to new site on forearm. Application sites evaluated at 24 h and 48 h post-
application. Non-irritant and non-sensitizer.64

49 male and female subjects. HRIPT. Semi-occlusive patch (2 cm x 2 cm) containing ~ 2 ml hydrolyzed silk (average mw = 1,000 Da; concentration not
stated) applied to back repeatedly during induction. Challenge patch applied for 24 h to new site. Application sites evaluated at 24 h and 48 h post-
application. Transient erythema (non-irritant and non-allergic in nature) observed during induction; 1 subject with cumulative skin irritation reaction after
removal of 9th induction patch. Subject also had barely perceptible erythema at challenge site (48-h reading). No clinically significant irritation or
evidence of allergic contact dermatitis.87
48 subjects. HRIPT. During induction, 20% aqueous hydrolyzed silk (20 µl on occlusive patch [8 mm diameter Finn chamber; 40 mm2 surface]) applied
for 48 h to the back repeatedly. 48-h challenge patch applied to new site on back. Non-irritant and non-sensitizer.16,88

Silk Powder

30 male and female subjects. Silk powder (0.05 g) applied for 48 h, under a closed patch, to the arm of each subject. Examinations for dermal reactions
after 1 h and 24 h. No skin irritation.43
 Table 5. Skin Irritation/Sensitization Potential of Silk Ingredients

Skin Irritation and Sensitization - In Vitro

Hydrolyzed Silk
The Irritection® assay. In vitro system that involves use of a proprietary solution containing both proteins and macromolecules in a well that is covered by
a membrane. Hydrolyzed silk applied to the Irritection® system at dose volumes of 25 µl, 50 µl, 75 µl, 100 µl, and 125 µl. Irritation measured
quantitatively using a spectrophotometer. Non-irritant (Ocular Irritection® scores: 0.25 to 0.40).70

EpiDermTM model assay. Test material (30 µl of aqueous solution containing 27% to 32% hydrolyzed silk) applied to human epidermal-derived
keratinocytes (cultured to form a multilayer). Non-irritant.71
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