Minoxidil en Cosmeticos Fda
Minoxidil en Cosmeticos Fda
Minoxidil en Cosmeticos Fda
FINAL REPORT
Contract Number
HHSF223201810176P
Submitted to:
Center for Food Safety and Applied Nutrition (CFSAN)
Office of Cosmetics and Colors
U.S. Food and Drug Administration
College Park, MD 20740-3835
Submitted by:
Department of Dermatology
Columbia University Medical Center
New York, NY 10032
September 2022
FDA-CU Final
Table of Contents
1 EXECUTIVE SUMMARY ...................................................................................................................8
2 PROJECT INFORMATION (Table 1) ................................................................................................9
3 PROJECT TIMELINE (Table 2) .......................................................................................................10
4 PROJECT 1: Alopecia Assessment of Test Hair Care Products Using Synchronized Murine HF
Model ......................................................................................................................................................11
4.1 Project Rationale and Objectives .............................................................................................11
4.2 Test Products ...........................................................................................................................11
4.3 Testing model and Methods .....................................................................................................12
4.4 Administration of Test Products................................................................................................13
4.5 Study Design ............................................................................................................................14
4.6 Tissue Collection and Storage..................................................................................................16
4.7 Analyses ...................................................................................................................................17
4.8 Results and Discussion ............................................................................................................19
4.8.1 Alopecia assessment of the test products ......................................................................... 19
4.8.1.1 Evaluation of DevaCurl Low-Poo Delight Cleanser (Exp-1) .......................................... 19
4.8.1.2 Evaluation of Aquaphor Baby Wash & Shampoo, Monat Renew Shampoo, and WEN
Sweet Almond Mint Cleansing Conditioner (Exp-2) ..................................................................... 32
4.8.1.3 Comparison between the mock cohorts in Exp-1 and Exp-2 ......................................... 44
4.8.2 Mast cells infiltration and distribution.................................................................................45
4.8.2.1 Evaluation of DevaCurl Low-Poo Delight Cleanser (Exp-1) .......................................... 45
4.8.2.2 Evaluation of Aquaphor Baby Wash & Shampoo, Monat Renew Shampoo, and WEN
Sweet Almond Mint Cleansing Conditioner (Exp-2) ..................................................................... 46
4.8.2.3 Validation of toluidine blue method ................................................................................ 46
4.8.2.4 Conclusion .....................................................................................................................46
4.8.3 Macrophage Evaluation.....................................................................................................54
4.8.3.1 Evaluation of DevaCurl Low-Poo Delight Cleanser (Exp-1), and Aquaphor Baby Wash &
Shampoo, Monat Renew Shampoo, and WEN Sweet Almond Mint Cleansing Conditioner (Exp-2)
54
4.8.3.2 Conclusion .....................................................................................................................55
4.8.4 Assessment of hair damage and structural abnormality ................................................... 58
4.8.5 Histopathological evaluation of liver ..................................................................................62
4.8.6 Body weight measurement ................................................................................................64
4.9 Summary and Conclusion ........................................................................................................66
4.10 Potential limitations and Recommendations............................................................................. 67
5 PROJECT 2: In vitro cytotoxicity assessments of ingredients found in hair care products. ............ 68
5.1 Project Rationale and Objectives .............................................................................................68
2
FDA-CU Final
3
FDA-CU Final
List of Figures
In vivo Studies
Figure 1. Materials for treatment and cleansing.
Figure 2. Study overview.
Figure 3. Image acquisition and measurement of skin pigmentation.
Figure 4. Visual assessment of skin pigmentation and hair growth from Day 0 (D0) to Day 84 (D84)
after treatment.
Figure 5. Mean gray intensity values of depilated areas quantified using ImageJ.
Figure 6. HF analysis at the anagen─telogen transition (Exp-1, Timepoint-1).
Figure 7. Dystrophic catagen and telogen in CYP-treated mice.
Figure 8. HF analysis at the telogen-anagen transition (Exp-1, Timepoint-2).
Figure 9. Visual assessment of skin pigmentation and hair growth from Day 0 (D0) to Day 98 (D98)
after treatment.
Figure 10. Mean gray values of depilated areas quantified using ImageJ.
Figure 11. HF analysis at the anagen-telogen transition (Exp-2, Timepoint-1).
Figure 12. HF analysis at the telogen-anagen transition (Exp-2, Timepoint-2).
Figure 13. Mast cell infiltration and degranulation at the anagen-telogen transition (Exp-1, Timepoint-
1).
Figure 14. Mast cell infiltration and degranulation at the telogen-anagen transition (Exp-1, Timepoint-
2).
Figure 15. Mast cell infiltration and degranulation at the anagen-telogen transition (Exp-2, Timepoint-
1).
Figure 16. Mast cell infiltration and degranulation at the telogen-anagen transition (Exp-2, Timepoint-
2).
Figure 17. Detection of mast cells by toluidine blue staining (A) and immunohistochemical staining
using mast cell tryptase antibody (B).
Figure 18. Immunohistochemical detection of macrophage (Exp-1, Timepoint-1).
Figure 19. Immunohistochemical detection of macrophage (Exp-1, Timepoint-2).
Figure 20. Immunohistochemical detection of macrophage (Exp-2, Timepoint-1).
Figure 21. Immunohistochemical detection of macrophage (Exp-2, Timepoint-2).
Figure 22. Hair shaft defects at the anagen-telogen transition (Exp-1, Timepoint-1).
Figure 23. Hair shaft defects at the telogen-anagen transition (Exp-1, Timepoint-2).
Figure 24. Hair shaft defects at the anagen-telogen transition (Exp-2, Timepoint-1).
Figure 25. Hair shaft defects at the telogen-anagen transition (Exp-2, Timepoint-2).
Figure 26. Histopathological evaluation of liver.
In vitro Studies
Figure 27. Cytotoxicity assessment of minoxidil and cisplatin in DPCs (A) and NHEKs (B)
Figure 28. Cytotoxicity assessment of Monat Renew shampoo (MO) in DPCs (A) and NHEKs (B)
Figure 29. Cytotoxicity assessment of WEN Sweet Almond Mint Cleansing Conditioner (WEN) in DPCs
(A) and NHEKs (B)
Figure 30. Cytotoxicity assessment of DevaCurl Low-Poo Delight Cleanser (DC) in DPCs (A) and
NHEKs (B)
4
FDA-CU Final
Figure 31. Cytotoxicity assessment of Aquaphor Baby Wash & Shampoo (AQ) in DPCs (A) and
NHEKs (B)
Figure 32. Cytotoxicity assessment of Acetyl tetrapeptide-3 in DPCs (A) and NHEKs (B)
Figure 33. Cytotoxicity assessment of Calendula extract in DPCs (A) and NHEKs (B)
Figure 34. Cytotoxicity assessment of Pequi oil in DPCs (A) and NHEKs (B)
Figure 35. Cytotoxicity assessment of CATC in DPCs (A) and NHEKs (B)
Figure 36. Cytotoxicity assessment of Lemon peel oil in DPCs (A) and NHEKs (B)
Figure 37. Cytotoxicity assessment of CAPB in DPCs (A) and NHEKs (B)
Figure 38. Cytotoxicity assessment of Coconut oil in DPCs (A) and NHEKs (B)
Figure 39. Cytotoxicity assessment of Dextran 40 in DPCs (A) and NHEKs (B)
Figure 40. Cytotoxicity assessment of Dextran 70 in DPCs (A) and NHEKs (B)
Figure 41. Cytotoxicity assessment of Guar in DPCs (A) and NHEKs (B)
Figure 42. Cytotoxicity assessment of Sunflower seed oil in DPCs (A) and NHEKs (B)
Figure 43. Cytotoxicity assessment of Lavender oil in DPCs (A) and NHEKs (B)
Figure 44. Cytotoxicity assessment of MCI in DPCs (A) and NHEKs (B)
Figure 45. Cytotoxicity assessment of MI in DPCs (A) and NHEKs (B)
Figure 46. Cytotoxicity assessment of Pea extract in DPCs (A) and NHEKs (B)
Figure 47. Cytotoxicity assessment of Polysorbate 60 in DPCs (A) and NHEKs (B)
Figure 48. Cytotoxicity assessment of Rosemary leaf extract in DPCs (A) and NHEKs (B)
Figure 49. Cytotoxicity assessment of Tomato seed oil in DPCs (A) and NHEKs (B)
Figure 50. Cytotoxicity assessment of Red clover extract in DPCs (A) and NHEKs (B)
Figure 51. Cytotoxicity assessment of Olus oil in DPCs (A) and NHEKs (B)
List of Tables
In vivo Studies
Table 1. Project information.
Table 2. Project timeline.
Table 3. Test Products and control drugs/products.
Table 4. Treatment cohorts.
Table 5. Data Collected.
Table 6. Mean gray values of depilated areas (Exp-1).
Table 7. % area of full hair growth at Day 84.
Table 8. % area with depigmented hairs at Day 84.
Table 9. % anagen skin at Day 84.
Table 10. % telogen skin at Day 84.
Table 11. HF analysis at Day 21 (Exp-1, Timepoint-1).
Table 12. HF analysis at Day 85 (Exp-1, Timepoint-2).
Table 13. Mean gray values of depilated areas (Exp-2).
Table 14. % area of full hair growth at Day 98.
Table 15. % anagen skin at Day 98.
Table 16. % telogen skin at Day 98.
Table 17. HF analysis at Day 21 (Exp-2, Timepoint-1).
Table 18. HF analysis at Day 98 (Exp-2, Timepoint-2).
5
FDA-CU Final
In vitro Studies
Table 32. Test Products
Table 33. Test ingredients and controls.
Table 34. The presence of the test ingredients in the four selected products.
Table 35. Experimental design for MTS assay.
Table 36. Viability (%) of DPCs treated with Monat Renew shampoo.
Table 37. Viability (%) of NHEKs treated with Monat Renew shampoo.
Table 38. Viability (%) of DPCs treated with WEN Sweet almond mint cleansing conditioner.
Table 39. Viability (%) of NHEKs treated with WEN Sweet almond mint cleansing conditioner.
Table 40. Viability (%) of DPCs treated with DevaCurl Low-Poo Delight Cleanser.
Table 41. Viability (%) of NHEKs treated with DevaCurl Low-Poo Delight Cleanser.
Table 42. Viability (%) of DPCs treated with Aquaphor Baby Wash & Shampoo.
Table 43. Viability (%) of NHEKs treated with Aquaphor Baby Wash & Shampoo.
Table 44. Viability (%) of DPCs treated with Acetyl tetrapeptide-3.
Table 45. Viability (%) of NHEKs treated with Acetyl tetrapeptide-3.
Table 46. Viability (%) of DPCs treated with Calendula extract.
Table 47. Viability (%) of NHEKs treated with Calendula extract.
Table 48. Viability (%) of DPCs treated with Pequi oil.
Table 49. Viability (%) of NHEKs treated with Pequi oil.
Table 50. Viability (%) of DPCs treated with CATC.
Table 51. Viability (%) of NHEKs treated with CATC.
Table 52. Viability (%) of DPCs treated with Lemon peel oil.
Table 53. Viability (%) of NHEKs treated with Lemon peel oil.
Table 54. Viability (%) of DPCs treated with CAPB.
Table 55. Viability (%) of NHEKs treated with CAPB.
Table 56. Viability (%) of DPCs treated with Coconut oil
Table 57. Viability (%) of NHEKs treated with Coconut oil.
Table 58. Viability (%) of DPCs treated with Dextran 40.
Table 59. Viability (%) of NHEKs treated with Dextran 40.
Table 60. Viability (%) of DPCs treated with Dextran 70.
6
FDA-CU Final
7
FDA-CU Final
1 EXECUTIVE SUMMARY
This final report provides an overview of preclinical investigations undertaken to evaluate hair loss
(alopecia) and the potential mechanisms of alopecia associated with the use of select commercially
available hair care products. The study consisted of two projects; Project 1, the in vivo study using a
murine model and Project 2, the in vitro cytotoxicity of test products and ingredients found in these hair
care products. A list of priority hair care products and ingredients was provided by the FDA and
included four test products and 21 test ingredients.
These investigations were conducted in stages. The initial stage comprised a series of in vivo pilot
studies to identify (i) variables and confounding factors that might affect the validity and results of the
experiment, (ii) the experimental conditions that minimally interfere with HF cycling, and (iii) an
application method as close as possible to the real “in use” situation to enhance the skin accessibility of
the test products. The results of these pilot studies helped establish the technical standards and
methods, validate the preclinical models used in Project 1, and test the technical feasibility of long-term
in vivo alopecia studies, for which there exists no extensive literature to date. In vitro pilot studies were
conducted to optimize experimental conditions and determine dose-ranges.
In Project 1, the potential association between the four hair care products and alopecia was evaluated
employing the depilation-induced synchronized hair follicle (HF) model in C57BL/6J mice. In Project 2,
the potential effects of the test products and the hair care product ingredients on cell viability and
growth were investigated using human hair follicle (HF) dermal papilla cells (DPCs) and normal human
epithelial keratinocytes (NHEKs). Test ingredients that demonstrated cytotoxicity were further evaluated
for apoptosis induction in DPCs.
This report is intended to highlight the design and development of appropriate research approaches
and to discuss the research findings. It is organized into two main parts: the first describes the results of
Project 1, and the second describes those of Project 2. Each of the projects includes a detailed
description of its testing models and methodologies, discusses its challenges, and provides
recommendations for future studies.
Key findings Project 1. Alopecia Assessment of Test Hair Care Products Using Synchronized
Murine HF Model.
• A delay in progression to the 2nd anagen phase was observed in mice treated with WEN Sweet
Almond Mint Cleansing Conditioner (WEN) or DevaCurl Low-Poo Delight Cleanser (DevaCurl),
compared to the mock cohort.
• Treatment with either Monat Renew Shampoo (Monat) or WEN substantially increased total
mast cell numbers at Day 98.
• WEN caused significant increases in mast cell activation.
Key findings Project 2. In vitro Cytotoxicity Assessments of Ingredients Found in Hair Care
Products.
• All test products demonstrated cytotoxicity within 24h in DPCs.
• Compared to the other test products, Monat was the most cytotoxic in both DPCs and NHEKs.
• Of the 20 test ingredients, 11 demonstrated varying degrees of cytotoxicity in DPCs within 72h.
Of these, 7 induced acute cytotoxicity in DPCs, decreasing cell viability by more than 50% within
24h. These included MCI, MI, CAPB, Lavender oil, Polysorbate 60, CATC, and Pea extract.
• Of all ingredients tested, MCI and MI were the most cytotoxic.
• Apoptosis was detected in DPCs treated with Guar, Lavender oil, or Rosemary extract, while
Calendula extract, CATC, and Pea extract primarily induced necrosis.
8
FDA-CU Final
Performance
Site
9
FDA-CU Final
* In response to the COVID-19 pandemic, Columbia University implemented a ramp-down of all non-
essential on-site laboratory research. Under the Institute of Comparative Medicine’s contingency plans,
the on-site research activity of Project 1 was deemed non-essential, as C57BL/6 mice used in this
study were commercially available and this research activity was not needed for a pending publication
or grant application. Therefore, Project-1 Exp-1, which was scheduled to begin on March 17, 2020,
was suspended, and mice were euthanized, causing a delay in the project's progress. Project
completion was further delayed due to the lack of research personnel and the Institutional hiring
freeze.
10
FDA-CU Final
11
FDA-CU Final
The mouse is an excellent model with which to study the hair cycle for several reasons: the first two
cycles of the mouse HFs are synchronized, the mouse hair cycle is short (~3 weeks), and the HF
stages have been well characterized and can easily be examined at specific time points in the cycle. In
addition, various transgenic murine models of hair abnormalities are available for studying the genetic
aspects of hair disorders [12, 13]. However, although mouse HFs share the same essential features as
human HFs, and HF cycling does not differ structurally between mice and humans, there exist some
species-specific differences. For example, the human hair cycle occurs asynchronously in the scalp,
and the anagen phase of human HFs lasts from 3−5 years [13]. Furthermore, the capacity for
percutaneous absorption likely differs between humans and mice, as the human dermis is substantially
thicker than the mouse dermis and contains fewer HFs. Moreover, mice do not suffer from androgenetic
alopecia (AGA), the most common form of hair loss in humans, and the key mechanisms controlling
androgen-dependent HF miniaturization in the human scalp are not recapitulated in mice [14]. These
species-specific differences in HF growth and regulation must be considered carefully when interpreting
the outcomes of mouse studies [14, 15].
This study used the inbred C57BL/6 strain, one of the most extensively studied and best-standardized
hair research models [16, 17]. In C57BL/6 mice, and other murine strains (e.g., CBA/J, C3H, BALB/c),
HFs on dorsal skin at postnatal day 60 (P60) are predominantly in the telogen stage. The removal of
telogen hair shafts by depilation immediately initiates synchronized hair growth with all follicles entering
the final stage of the growth cycle (anagen VI) on day 9 post depilation. After full anagen development,
the consecutive stages, catagen, and telogen, develop spontaneously in a relatively homogeneous
pattern. The depilation-induced synchronized HF model is widely used in hair biology research as it
allows the evaluation of specific HF stages at specific time points. It also provides an adequate in vivo
platform for the preclinical evaluation of both drug efficacy and safety testing for humans [13].
Depilation
To induce synchronized hair growth, the back hairs of P60 mice were shaved using an animal clipper.
Nair hair removal cream (Lot no. LL8331, purchased from Amazon) was then applied to the shaved
dorsal skin for 3 min to remove the hair shafts. The depilated area was thoroughly washed using a
spray of warm water. Depilation was performed under anesthesia.
12
FDA-CU Final
Housing condition
Mice were housed in groups of four animals per cage under pathogen-free conditions in the animal
facilities of Columbia University. Mice were kept in 12h light/dark cycles in a temperature-controlled
(20–25ºC) room with a 50~60% relative humidity and given a standard rodent diet and water ad libitum.
Ethics statement
All animal experiments described in this report and animal procedures including euthanization were
performed according to the approved Columbia University Institutional Animal Care and Use Committee
(IACUC) protocol (AC-AABM0551).
Figure 1. Materials for treatment and cleansing. A. Electric heating pad, B. Paper towels, C. Cotton
pads, D. Test product in a 50 ml conical tube, E. 1 ml syringe, F. Water bottle, G. Baby bottle warmer,
H. Soap trays, I. Isoflurane machine, J. Anesthesia induction chamber, and K. Timer.
13
FDA-CU Final
Application
The depilated dorsal site was first wetted using a water-soaked cotton pad. The test product was
applied to the site using a repeating pipette (Bel-Art SP Scienceware). The site was then gently rubbed
and left uncovered for 10 min. After 10 min, the site was washed with a spray of warm water. The entire
procedure typically took ~ 15 min per mouse and was performed under anesthesia in conjunction with
electric heating pads to prevent hypothermia.
Administration volume
0.3 ml (0.05 ml/cm2) of the test product was applied per mouse. This amount sufficiently covers the
entire application site (~ 6 cm2) and is comparable to that recommended for the WEN products (0.04 ml
─ 0.07 ml/cm2 in humans with short hair). The administration volume was increased to 0.5 ml per
application when hairs in the depilated area regrew.
CYP-induced disruption of actively growing anagen HFs in C57BL/6 mice is a clinically relevant model
that has been extensively used in studying the biology of chemotherapy-induced alopecia [20-22].
Minoxidil (2,4-diamino-6-piperidino-pyrimidine-3-oxide) is the most commonly used drug for the
treatment of androgenetic alopecia. It has been shown to shorten the telogen stage, while prolonging
the anagen stage through both proliferative and anti-apoptotic effects on the dermal papilla cells of
human HFs [23]. A topical minoxidil solution (2-5%) has also been shown to enhance hair growth in
mice (e.g., C57BL/6J, CBA/J mice) [24, 25].
Cohorts
The animal study was performed in two sequential experiments, consisting of nine cohorts of 16 mice
each (Table 4). Baseline body weight and baseline blood were obtained from all mice. The treatment
began two days after depilation. The first experiment (Exp-1, Cohorts 1-5) included nontreated, mock,
DevaCurl, Rogaine, and CYP. Exp-2 (Cohorts 6-9) included mock, Aquaphor, Monat, and WEN.
Nontreated and CYP cohorts served to validate normal HF cycling in C57BL/6 mice. The mock cohorts
of Exp-1 and Exp-2 received water and were subjected to the same experimental conditions as the
mice that received the test products. The mock cohorts served as a bridge between the two
experiments.
14
FDA-CU Final
Timepoints
Half of the mice in each cohort (n=8) were evaluated at the 1st anagen─1st telogen HF transition
(Timepoint 1) and the remaining half (n=8) at the 1st telogen─2nd anagen HF transition (Timepoint 2)
(Fig. 2A). The anagen-telogen HF transition corresponded to Day 21, and the telogen-anagen HF
transition corresponded to Day 85 (Exp-1) and Day 98 (Exp-2).
Figure 2. Study overview. A. Treatment timeline. B. Schematic representation of hair cycle associated
changes in HF length and size (diameter, arrows) in correlation with the panniculus carnosus (PC) and
15
FDA-CU Final
the dermis/subcutis border [1]. A, anagen; C, catagen; T, telogen. C. Representative image of mouse
skin showing different layers of skin and HFs. HF length and size (diameter) measured in this study are
indicated. Blue dotted line, epidermis/dermis border; black dotted line, dermis/subcutis border; red
dotted line, subcutis/PC border; solid yellow line, HF diameter. D. Representative images of
nondegranulated (nonactivated) mast cells (black arrows) and degranulated (activated) mast cells with
extracellular granules (magenta arrows). Scale bar = 50 µm.
Data collection
Data were collected for the following evaluations (Table 5): i) the hair growth pattern and presence of
alopecia (Evaluation 1), ii) the correlations between skin pigmentation and HF morphology (Evaluation
2), iii) the extent of hair damage (Evaluation 3), and iv) the presence of mast cells (Evaluation 4) and
macrophages (Evaluation 5).
Body weight measurements and liver histology were assessed for possible systemic effects of the test
products (Evaluation 6).
Skin: Full-thickness dorsal skin (1 x 2 cm) was collected at timepoints 1 and 2 from the same area on
all mice. A longitudinal section was fixed in 10% buffered formalin and paraffin-embedded for
histological analyses. A portion was snap-frozen and stored at -80 °C for future studies.
16
FDA-CU Final
Hair: Hair samples were obtained at timepoints 1 and 2 by plucking them lightly from the unshaved
location on the dorsal skin. The collected hairs were placed in a microcentrifuge tube and stored at
room temperature.
Liver: Liver samples were collected at timepoints 1 and 2. One piece of liver tissue (1 x 1 cm) was
taken from the right lobe, formalin-fixed, and paraffin-embedded for histological evaluation.
4.7 Analyses
Body weight measurement
Mouse body weight was obtained at baseline and every week.
Photo documentation
Images of mouse dorsal skin were acquired twice a week during anagen progression, and once a week
during the telogen phase. A light-equipped photo box that provides consistent lighting and prevents
shadow and reflection in the photo was used for photo documentation (Fig. 3A). The distance of the
light source to the subject (23 cm) was kept constant for all imaging.
Figure 3. Image acquisition and measurement of skin pigmentation. A. Photo box used in study. B.
Measurement of gray values (a) and % gray area (b).
17
FDA-CU Final
Immunohistochemistry
Immunohistochemical staining was performed on 5-µm thick paraffin-embedded skin sections.
Following deparaffinization and rehydration through xylene and graded alcohols, sections were boiled
in citric acid antigen unmasking solution (Vector Laboratories). Endogenous peroxidase activity was
quenched by incubating sections for 10 min in 3% hydrogen peroxide solution. Staining was performed
using ImmPRESS® Horse Anti-Rabbit IgG PLUS Polymer Kit and ImmPACT (TM) DAB HRP Substrate
(Vector Laboratories) according to the manufacturer’s protocols. Staining for mast cells was carried out
using Mast Cell Tryptase Rabbit anti-Human/Mouse/Rat antibody (1:200 dilution, clone ARC2328,
Invitrogen), and F4/80 Rabbit anti-Mouse antibody (1:250 dilution, clone SP115, Invitrogen) was used
to stain macrophages. Sections were imaged at 40x magnification and evaluated using the Aperio AT2
DX System and Aperio ImageScope software, respectively, as described in the previous section. Five
sections per cohort were evaluated for mast cells. Four to five mice per cohort (at least 3 – 4 mm2 total
tissue area per mouse) were evaluated for F4/80+ macrophages.
18
FDA-CU Final
Statistical methods
Statistical analysis was performed using GraphPad (version 9.4.1.681, GraphPad Software, Inc.). Data
were analyzed using two-way ANOVA with Bonferroni multiple comparison test. Data are presented as
mean ± SD. Adjusted p values are included in the tables. p<0.05 was considered statistically
significant.
Skin pigmentation and hair recovery: Melanogenic truncal skin melanocytes in pigmented mice are
confined to HFs, where they become melanogenically active during the anagen III phase of the hair
growth cycle and are directly involved in hair shaft pigmentation. As no melanin is synthesized in
telogen skin, changes in skin pigmentation from unpigmented (pink) to pigmented (gray to black)
indicate active hair growth [30-32], which can be tracked and measured during the entire treatment
period and correlated with hair growth pattern.
HF morphology and stages of HF growth: HF disorders and abnormalities in HF cycling that affect the
duration of the anagen and telogen phases are key mechanisms underlying the pathogenesis of
alopecia. From a clinical perspective, both premature termination of the anagen phase, as seen in
androgenetic alopecia (AGA), and premature entry into the telogen phase, increase the percentage of
HFs in the telogen phase [33]. In AGA, HF miniaturization is known to impair hair growth in the anagen
phase and lead to a shortening of this phase, thereby prolonging the telogen phase [34]. A prolonged
telogen phase that delays the onset of anagen has also been observed in mice in response to the
topical application of glucocorticoids, prototypic stress hormones, or estrogen [33]. HF morphology and
stages of HF growth were assessed in H&E-stained longitudinal skin sections.
HFs maintain their maximal lengths between the anagen VI and catagen II phases. During this time, the
dermal papilla is located close to the panniculus carnosus, and anagen and catagen HFs are not easily
distinguishable using morphologic criteria and a light microscope [19]. Therefore, catagen I-II HFs were
excluded from the analysis.
HF size and length: HF lengths increase during anagen phases I–VI and decrease during catagen
phases I–VIII. These hair cycle-associated fluctuations in HF lengths correlate with changes in skin
thickness [19]. Therefore, HF sizes and lengths were measured as additional parameters of HF cycling.
In nontreated mice, the hair cycle progressed to the catagen/telogen stage, entering the 1st telogen
stage by Day 24, (Figs. 4A, 5). The HFs remained in telogen for about 4-5 weeks before transitioning
to the second anagen stage at about Day 57, with all animals having fully regrown their hair by Day 77.
19
FDA-CU Final
In some animals, spots of pink skin were noticeable at about this time (Fig. 4A), suggesting a transition
to the 2nd telogen stage. At Day 84, the mean gray value was similar to that at Day 77 (Fig. 5, Table 6),
and 99.02% of the depilated areas displayed a normal hair coat (Fig. 4A, Table 7).
A single administration of CYP at the onset of the 1st anagen stage significantly impaired hair growth.
While the mice eventually recovered and regrew their hair, achieving 93.02% hair recovery at Day 84
(Fig. 4B, Table 7), 36.54% of the hairs that regrew in the depilated areas were depigmented and
displayed a rough texture (Table 8).
Compared to the nontreated cohort, the mice treated with water and exposed to the same experimental
conditions (mock) as those in the DevaCurl and Rogaine cohorts showed a delay in HF cycling. The
transition to catagen/telogen occurred at about Day 30, followed by entry into the second anagen stage
around Day 60 (Fig. 5, Table 6), and reaching 77.63% hair growth at Day 84 (Fig. 4B, Table 7).
Minoxidil (Rogaine) has been shown to shorten telogen, causing premature entry of resting HFs into
anagen. Consistent with the stimulatory effect of minoxidil on HFs, in mice treated with Rogaine, the
progression to the anagen stage was accelerated after Day 60 (Fig. 5, Table 6). At Day 84, the extent
of hair recovery in this cohort was significantly higher than in the mock (99.26% vs. 77.63% in mock)
and was comparable to that in the nontreated mice (vs. 99.02% in nontreated) (Fig. 4E, Table 7).
In contrast, a significant delay in HF cycling was observed in mice treated with DevaCurl, resulting in
56.11% hair recovery at Day 84 (p=0.0034 vs. mock) (Fig. 4D, Table 7). While four mice in the mock
cohort fully regrew their hair, a full coat was visible only in one mouse in the DevaCurl cohort (Fig. 4D).
The area of gray skin, which represents the active growth phase, was smaller in the DevaCurl cohort
than in the mock control (Table 9). Notably, the percent area of pink telogen skin in the DevaCurl
cohort was higher than that in the mock cohort (16.68% vs. 1.21% in mock) (Table 10). However, these
data were not statistically significant.
HF morphology
Because premature entry into the telogen phase contributes to hair loss, telogen HFs were quantified in
skin sections at the anagen─telogen transition. Consistent with gray skin color, the HFs at Day 21 were
predominantly in the catagen stage in all cohorts, and albeit not statistically significant, small fractions
of HFs in the nontreated (5.77%) and Rogaine (10.76%) cohorts progressed to the telogen phase (Fig.
6A, 6B; Table 11).
CYP has been shown to prematurely induce the catagen phase and increase telogen HFs [35].
Furthermore, a higher dose of CYP (150 mg/kg, the same dose used in this study) has been shown to
induce dystrophic catagen and telogen in C57BL/6 mice [16]. In this experiment, the percentage of
telogen HFs was substantially higher in the CYP cohort (29.65% vs. 5.77% in nontreated, p=0.005)
(Fig. 6B, Table 11), and although not present in every HF, some features of dystrophic catagen and
telogen were detectable (e.g., ectopic melanin clumps, abnormal widening of the hair canal, remnants
of the hair shaft) (Fig. 7). Given the relatively synchronous hair cycling at Day 21, we found no
substantial differences in HF size or length among different cohorts (Fig. 6C, 6D).
At Day 85, hair recovery in the CYP and Rogaine cohorts was similar to that observed in nontreated
mice (99.02% in nontreated, 93.02% in CYP, and 99.26% in Rogaine). It is important to note that
mouse club hairs from the telogen phase and growing hairs typically share a hair follicle, and that
healthy murine HFs often retain old hair shafts from preceding cycles [36]. For these reasons, hair loss
may not be readily detectable by visual assessment. This is likely the case with these cohorts, as
evidenced by the predominance of HFs in the telogen phase in haired skin sections on these mice,
indicating progression to the 2nd telogen phase (Fig. 8A, 8B; Table 12). Compared to the nontreated
20
FDA-CU Final
cohort, a delay in HF cycling was observed in the mock cohort, in which 30.71% of HFs were in the 2nd
anagen phase and 60% were in the 2nd catagen phase. Consistent with the delayed hair recovery
observed in mice treated with DevaCurl, the majority of DevaCurl HFs were distributed between the 1st
telogen and 2nd anagen phases. Although 47.06% of HFs entered the 2nd anagen phase─corroborated
by larger HF sizes and lengths (Fig. 8C, 8D; Table 12), 46.39% of DevaCurl HFs remained in the 1st
telogen phase at Day 85 (Fig. 8B, Table 12). Although the differences in the 1st telogen phase and the
2nd anagen phase were not statistically significant, the percentage of HFs in the 2nd catagen phase was
significantly lower in mice treated with DevaCurl (6.55% vs. 60% in Mock, p=0.0173), suggesting
overall delay in the telogen─anagen transition.
21
FDA-CU Final
22
FDA-CU Final
23
FDA-CU Final
24
FDA-CU Final
25
FDA-CU Final
Figure 4. Visual assessment of skin pigmentation and hair growth from Day 0 (D0) to Day 84
(D84) after treatment. D0−D20: 16 mice per cohort. D24–D84: 8 mice per cohort. At D21, 8 mice from
each cohort were euthanized and tissue samples were collected for analyses.
26
FDA-CU Final
Figure 5. Mean gray intensity values of depilated areas quantified using ImageJ. Refer to Table 6
for the adjusted (adj.) p values for all data points.
27
FDA-CU Final
28
FDA-CU Final
C
CYP
T
C
C C
Mock
C
DevaCurl
C
Rogaine
T
T
100 60
*
Avg HF Diameter (µm)
600
Avg HF length (µm)
50
80
40
% HF
60 400
* 30
40
20 200
20 10
0 0 0
NT CYP Mock DC RO NT CYP Mock DC RO NT CYP Mock DC RO
29
FDA-CU Final
T
Nontreated
C C
100 µm 200 µm
C
CYP
SG T SG
T C
100 µm 50 µm 50 µm
Figure 7. Dystrophic catagen and telogen in CYP-treated mice. Representative images of H&E-
stained longitudinal sections of mouse dorsal skin. CYP, cyclophosphamide; C, catagen HF; T, telogen
HF; SG, sebaceous gland; yellow arrows, melanin clumps; blue arrows, abnormal widening of hair
canal; red arrows, remnants of the hair shaft. Images are presented at multiple magnifications for better
visualization.
30
FDA-CU Final
B. % HF Stage
NT 1st T 1st T 2nd A 2nd C 2nd T
80 140%
CYP
Mock
70
60 120%
*
DC 50
RO 40 100%
30
20 80%
10
2nd T 0 2nd A 60%
40%
20% *
0%
NT CYP Mock DC RO
2nd C
31
FDA-CU Final
70
500 *
40 300
*
30
200
20
100
10
0 0
NT CYP Mock DC RO NT CYP Mock DC RO
4.8.1.2 Evaluation of Aquaphor Baby Wash & Shampoo, Monat Renew Shampoo, and WEN
Sweet Almond Mint Cleansing Conditioner (Exp-2)
32
FDA-CU Final
sweet Almond Mint Cleansing Conditioner were similar to those of the mock cohort (Figs. 9D & 10;
Tables 13 and 14). The hair cycle in the mock cohort progressed to catagen-telogen, entering the 1st
telogen stage around Day 24 and Day 28. The hair growth cycle in the mock cohort remained
predominantly in the telogen stage for about 7.5 weeks (from Day 24 to Day 77) and then progressed to
the 2nd anagen phase around Day 81, reaching 87.28% hair regrowth at Day 98 (Figs. 9A, 10; Tables
13 & 14). In contrast, the transition to the 2nd anagen stage was observed at around Day 42 in mice
treated with Monat (Fig. 9C), and the mean gray value was substantially higher in this cohort compared
to the mock cohort at Day 66 (210.83 Monat vs. 177.63 mock, p<0.0001) (Fig. 10, Table 13). A slight
increase in the mean gray value was also noticed in mice treated with Aquaphor at Day 66 (184.99
Aquaphor vs. 177.63 mock). However, these increases were statistically insignificant (Figs. 9B & 10,
Tables 13 & 14). WEN also caused a slight increase in the mean gray value at Day 66 (186.64 WEN
vs. 177.63 in mock, p=0.1673) (Fig. 10, Table 13). However, this increase was not statistically
significant, and the hair cycling pattern of the WEN-treated mice was comparable to that of the mock
mice (Fig. 9D).
At Day 98, the hair growth in mice treated with Aquaphor was comparable to that observed in the mock
mice (88.21% Aquaphor vs. 87.28% mock) (Fig. 9B, Table 14). Compared to the mock cohort, in mice
treated with Monat, the transition to the 2nd anagen stage was observed at around Day 42 with all
animals having fully regrown their hair by Day 77 (Figs. 9C, 10; Table 14). At Day 98, these mice
displayed 99.48% of hair recovery (vs. 87.28% in mock, p<0.05) in the depilated areas (Table 14).
In contrast, a substantial delay in HF cycling was observed in mice treated with WEN, resulting in
70.41% hair recovery at Day 98 (p=0.003) (Fig. 9D, Table 14). While four mice in the mock cohort fully
regrew their hair, a full coat was visible only in one mouse in the WEN cohort (Fig. 9D), and anagen
progression and hair growth were observed in 84.70% of the depilated areas in the WEN cohort at Day
98 (vs. 99.87% in mock, p=0.0086) (Table 15). While the mean gray values did not differ substantially
among cohorts (Fig. 10, Table 13), the pink telogen skin was observed in 15.30% of the depilated area
in the WEN cohort at Day 98 (vs. 0.13% mock, p=0.0086) (Table 16), indicating a delay in the 1st
telogen-to-2nd anagen transition. These results demonstrate hair cycle abnormalities in mice treated
with WEN and suggest that the impaired hair growth associated with WEN may involve a prolonged
duration of the telogen stage, thereby delaying anagen induction and subsequent hair growth.
HF morphology
At Day 21, HF cycling progression in the test cohorts (i.e., Aquaphor, Monat, WEN cohort) appeared to
be faster than that in the mock cohort (Fig. 11A, 11B; Table 17). Compared to the catagen phase
observed in the mock cohort, the test cohorts displayed higher proportions of telogen HFs and smaller
and shorter HFs (Fig. 11A-D, Table 17), suggesting progression to the subsequent telogen phase. At
Day 98, the % area of anagen skin and hair growth was >99% for the mock, Aquaphor, and Monat
cohorts, whereas 84.70% for the WEN cohort (Table 15). These data indicated that the majority of
animals in the mock, Aquaphor, and Monat cohorts had transitioned through the 2nd anagen phase and
regrown their hair, whereas, in the WEN cohort, a full coat was visible in only one mouse (Fig. 9D).
Consistent with this observation, the majority of HFs in Aquaphor (60%, p<0.0001) and Monat (96.12%,
p<0.0001) proceeded to the 2nd telogen phase (vs. 0% in mock), indicating accelerated hair growth in
these mice compared to the mock cohort (Fig. 12A, 12B; Table 18). In contrast, in the WEN cohort, the
telogen─anagen transition was significantly delayed. Compared to 97.37% of mock HFs that
progressed to the 2nd anagen, only 28.80% of HFs progressed to the 2nd anagen phase (p<0.0001) in
mice treated with WEN, while 71.20% (p<0.0001) remained in the 1st telogen phase (Table 18).
Compared to the mock cohort, the HF sizes and lengths were smaller in the Aquaphor, Monat, and
WEN cohorts due to the predominance of HFs in the telogen phase (Fig. 12C, 12D; Table 18).
33
FDA-CU Final
34
FDA-CU Final
35
FDA-CU Final
36
FDA-CU Final
37
FDA-CU Final
38
FDA-CU Final
Figure 9. Visual assessment of skin pigmentation and hair growth from Day 0 (D0) to Day 98
(D98) after treatment. Dorsal hairs were shaved at D20 to better observe changes in skin
pigmentation. One mouse in the Aquaphor cohort died at D26 (blue box). A WEN-treated mouse with
weight loss (red box); this mouse was included in the timepoint 1 and sacrificed at Day 21.
39
FDA-CU Final
150
100
50
0
D0 D3 D7 D10 D14 D17 D24 D28 D31 D35 D42 D49 D56 D59 D63 D66 D70 D77 D81 D84 D87 D91 D94 D98
Figure 10. Mean gray values of depilated areas quantified using Image. Refer to Table 13 for the
adjusted (adj.) p values for all data points. Gray value = pure white value (255) - gray value from
ImageJ.
40
FDA-CU Final
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis. Adjusted
(adj.) p values are included. Bold type: p < 0.05. Gray skin without visible hairs was excluded. % area
of full hair growth = {area of visible hair growth ÷ total depilated area} x 100.
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis. Adjusted
(adj.) p values are included. % area of anagen skin = {area of gray skin without visible hairs ÷ total
depilated area} x 100.
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis. Adjusted
(adj.) p values are included. Bold type: p < 0.05. % telogen skin = {area of pink skin without visible hairs
÷ total depilated area} x 100.
41
FDA-CU Final
Mock
C C C C C C
C
Aquaphor
T T T C
C C C C
T
Monat
T
C C C C C
T
WEN
C C C C
40 400
*
Avg HF length (µm)
80 *
% HF stage
30 300
60
20 200
40
10 100
20
0 0 0
Mock AQ MO WEN Mock AQ MO WEN Mock AQ MO WEN
42
FDA-CU Final
B. % HF Stage
Mock 1st T 1st T 2nd A 2nd C 2nd T
100
AQ 90 140%
MO 80
WEN
70
60
120% **
50
40 100% ** **
30
20
80%
2nd T
10
0 2nd A **
60% **
40%
20%
0%
2nd C Mock AQ MO WEN
43
FDA-CU Final
70
500
30
200
20
100
10
0 0
Mock AQ MO WEN Mock AQ MO WEN
The rates of hair cycling and subsequent hair recovery differed between the mock cohorts, with overall
delay observed in the Exp-2 mock cohort.
The Exp-2 mock cohort remained predominantly in the telogen stage for about 7.5 weeks before
transitioning to the 2nd anagen phase, as compared to the 5-6 weeks observed for the Exp-1 mock
cohort. Given the identical study design (e.g., experimental conditions, housing, researcher, etc.), the
reason for this discrepancy in HF cycling between the two mock cohorts is uncertain. As in humans,
mouse HF cycling can be influenced by various genetic and nongenetic conditions. For example,
mouse hair growth is highly susceptible to environmental factors, such as season, humidity, cage type,
and diet, which can cause stress in rodents, leading to various abnormal physiological responses,
including alopecia. Indeed, psychoemotional stress has been shown to alter HF cycling and
prematurely terminate the anagen stage in C57BL/6 mice [37]. Seasonal variation in response to
44
FDA-CU Final
stressful situations has also been reported in C57BL/6 mice [38], and a higher incidence of hair loss
and dermatitis in the winter has been observed in the mouse colony at Jackson Laboratory. In addition,
mice are sensitive to various scents, and certain chemosignals or pheromones have been shown to
invoke stress responses in mice [39]. Peppermint oil, menthol, and red clover can act as effective
mouse deterrents. Menthol is present in WEN Sweet almond mint cleansing conditioner and red clover
extract is one of the ingredients found in Monat Renew shampoo. The relevance of these confounding
variables and the extent of their effects on our study require further investigation. Nevertheless, the hair
cycle in the mock cohort eventually progressed to the 2nd anagen phase around Day 81, reaching
87.28% hair regrowth at Day 98 (Table 14). Despite this difference in the hair growth cycle, there were
no statistically significant differences in mock cohorts in body weight or skin pigmentation (Table 19).
Mast cells are crucial immunomodulatory cells that tend to be preferentially localized in certain skin
regions, such as the perivascular, perifollicular, and perineural dermis [40]. Mast cells release
proinflammatory mediators from cytoplasmic granules by degranulation, which is indicative of their
activation. Even in the absence of visible signs of skin inflammation, increases in the numbers of total
and activated mast cells are detectable in the dermis of non-scarring and non-inflammatory alopecia,
including telogen effluvium and alopecia areata (AA) [41], and in AA, perifollicular mast cells have been
implicated in facilitating cross-talk with CD8+ T cells [29].
Skin tissue sections were evaluated for the presence of mast cells using toluidine blue stain, one of the
most frequently used metachromatic stains for the detection of mast cells [42]. Infiltrating and
degranulating mast cells in the dermis and subcutis were quantified, as were degranulating perifollicular
and follicular mast cells.
The numbers of total and degranulated mast cells did not differ substantially among all cohorts at Day
21 (Fig. 13A, 13B; Table 20). However, the proportion of degranulated active mast cells was
significantly higher in the CYP and DevaCurl cohorts (35.88% in CYP vs. 18.41% in nontreated, p
<0.0001; 36.33% in DevaCurl vs. 26.71% in mock, p=0.0224) (Fig. 13C, Table 20). Furthermore, in the
CYP cohort, follicular mast cell activity was substantially higher (12.64% follicular vs. 3.41%
perifollicular) (Fig. 13D, 13E; Table 20). At Day 85, the Rogaine cohort showed a slight increase in the
total number of mast cells (75.28 in Rogaine vs. 53.6 in mock, p=0.0204) (Fig. 14A, 14B; Table 21).
Given Rogaine’s positive effects on hair growth, the reasons for this increase in mast cells are unclear.
In humans, chronic administration of Rogaine has been associated with several adverse cutaneous
effects, including scalp pruritus [43], a condition in which mast cells are considered the main conductors
of itch [44, 45]. Nevertheless, the relevance of mast cells to pruritus in this study and its link to Rogaine
warrant further investigations. Except for the Rogaine cohort, no significant differences in mast cell
activation and distribution were observed among all cohorts (Fig. 14C-E, Table 21).
45
FDA-CU Final
4.8.2.2 Evaluation of Aquaphor Baby Wash & Shampoo, Monat Renew Shampoo, and WEN
Sweet Almond Mint Cleansing Conditioner (Exp-2)
At Day 21, although the total number of mast cells was higher in Aquaphor and WEN cohorts (Fig. 15A,
15B; Table 22), no statistically significant differences were observed in mast cell activation and
distribution (Fig. 15C-E, Table 22). At Day 98, mice in the Monat and WEN cohorts showed substantial
increases in the total number of mast cells (63.02 in Monat, p<0.0001; 61.13 in WEN, p<0.0001 vs.
36.58 in mock) (Fig. 16A, 16B; Table 23). WEN also caused significant increases in mast cell
activation (44.66% in WEN vs. 23.36% in mock, p<0.0001) (Fig. 16C, Table 23).
Notably, mast cell activity appeared to be preferentially localized within the HFs in mice treated with
WEN, as the percentage of degranulated follicular mast cells was higher compared to those in the
mock cohort (7.80% in WEN vs. 0.25% mock) (Fig. 16A, 16C, 16D; Table 23). This difference,
however, was not statistically significant (Table 23).
Mast cell proteases, including carboxypeptidase, chymase, and tryptase, represent the major protein
components of secretory granules [42]. Immunohistochemical staining of select skin sections using
mast cell tryptase antibody (Clone ARC2328, Invitrogen) showed a level of mast cell detection similar to
that of metachromatic staining, validating the toluidine blue method used in this study (48.7±28 mast
cells/mm2 by immunohistochemical staining vs. 46.3±15.03 by toluidine blue staining, n=5 skin sections
from Exp-2, timepoint-2) (Fig. 17).
4.8.2.4 Conclusion
WEN caused significant increases in mast cell degranulation compared to the mock cohort at Day 98,
suggesting the possible involvement of mast cell activity in delayed progression to the anagen phase.
DevaCurl also increased mast cell activity; however, this increase was detectable only at the anagen-
telogen transition (Day 21).
46
FDA-CU Final
Nontreated
CYP
Mock
DevaCurl
Rogaine
B. C.
100 NT CYP Mock DC RO 60
degranulated mast cells/mm2
80 50
** *
40
60
30
40 * 20
20 10
0 0
Total Degranulated NT CYP Mock DC RO
47
FDA-CU Final
D. E.
NT CYP Mock DC RO NT CYP Mock DC RO
No. of degranulated perifollicular
% degranulated perifollicular
and follicular mast cells/mm2
10 20
*
6
10
4
5
2
0 0
Perifollicular Follicular Perifollicular Follicular
Figure 13. Mast cell infiltration and degranulation at the anagen-telogen transition (Exp-1,
Timepoint-1). A. Representative images of toluidine blue-stained longitudinal sections of mouse dorsal
skin. Red arrows, mast cells (MCs). B. Average number of infiltrating and degranulating MCs per mm2.
C. % degranulation. D. Average number of degranulating perifollicular and follicular MCs. E. %
degranulation of perifollicular and follicular MCs. The graph represents the mean ± SD. n = 4-7 mice
per cohort *, p < 0.05; **, p < 0.0001. Refer to Table 20 for the adjusted p values for all data points. NT,
nontreated; CYP, cyclophosphamide; DC, DevaCurl, RO, Rogaine. Scale bar = 200 µm.
Table 20. Mast cell infiltration and degranulation at Day 21 (Exp-1, Timepoint-1).
Total No. MC/ No. Degranulated MC/ % Degranulation No. Perifollicular No. Follicular MC/ % Perifollicular % Follicular
mm2 mm2 MC/ mm2 mm2
NT mean ± SD 20.1 ± 5.91 3.8 ± 2.55 18.41 ± 10.34 0.48 ± 0.35 0.55 ± 0.93 2.44 ± 2.31 2.88 ± 5.28
adj. p-value
CYP mean ± SD 26.18 ± 6.54 9.85 ± 5.21 35.88 ± 9.37 0.95 ± 0.62 3.4 ± 1.8 3.41 ± 1.63 12.64 ± 5.22
adj. p-value 0.6287 0.6467 <0.0001 >0.9999 >0.9999 >0.9999 0.0309
(vs. NT)
MOCK mean ± SD 29.44 ± 7.67 7.75 ± 2.51 26.71 ± 6.42 2.21 ± 1.09 0.05 ± 0.12 7.47 ± 2.73 0.18 ± 0.43
adj. p-value 0.0300 >0.9999 0.0818 >0.9999 >0.9999 >0.9999 >0.9999
(vs. NT)
DC mean ± SD 23.73 ± 4.52 8.77 ± 3.6 36.33 ± 10.42 2.23 ± 1.55 0.04 ± 0.11 8.71 ± 4.9 0.15 ± 0.37
adj. p-value 0.6736 >0.9999 0.0224 >0.9999 >0.9999 >0.9999 >0.9999
(vs. Mock)
RO mean ± SD 32.2 ± 9.03 10.34 ± 7.3 29.18 ± 15.24 2.29 ± 1.53 0±0 6.69 ± 3.94 0±0
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
(vs. Mock)
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis.
Adjusted (adj.) p values are included. Bold type: p < 0.05. % degranulated = (No. degranulated
MC ÷ Total No. of MC) x 100. MC, mast cells.
48
FDA-CU Final
Nontreated
CYP
Mock
DevaCurl
Rogaine
B. NT CYP Mock DC RO C.
100
degranulated mast cells/mm2
60
*
% degranulated mast cells
No. of infiltrating total or
80 50
40
60
30
40
20
20 10
0 0
Total Degranulated NT CYP Mock DC RO
49
FDA-CU Final
D. E.
No. of degranulated perifollicular NT CYP Mock DC RO NT CYP Mock DC RO
10 20
% degranulated perifollicular
and follicular mast cells/mm2
6
10
4
5
2
0 0
Perifollicular Follicular % Perifollicular % Follicular
Figure 14. Mast cell infiltration and degranulation at the telogen-anagen transition (Exp-1,
Timepoint-2). A. Representative images of toluidine blue-stained longitudinal sections of mouse dorsal
skin. Red arrows, Mast cells (MCs). B. Average number of infiltrating and degranulating MCs per mm2.
C. % degranulation. D. Average number of degranulating perifollicular and follicular MCs. E. %
degranulation of perifollicular and follicular MCs. The graph represents the mean ± SD. n = 4-7 mice
per cohort. *, p < 0.05. Refer to Table 21 for the adjusted (adj.) p values for all data points. NT,
nontreated; CYP, cyclophosphamide; DC, DevaCurl, RO, Rogaine. Scale bar = 100 µm.
Table 21. Mast cell infiltration and degranulation at Day 85 (Exp-1, Timepoint-2).
Total No. MC/ No. Degranulated MC/ No. Perifollicular MC/ No. Follicular MC/
% Degranulation % Perifollicular % Follicular
mm2 mm2 mm2 mm2
NT mean ± SD 66.13 ± 14.61 13.18 ± 6.68 19.5 ± 7.15 1.23 ± 0.82 0.42 ± 0.49 1.75 ± 0.75 0.57 ± 0.66
adj. p-value
CYP mean ± SD 58.36 ± 22.16 14.32 ± 5.7 24.56 ± 2.44 3.15 ± 2 0.91 ± 1.43 6.22 ± 4.37 1.25 ± 1.75
adj. p-value (vs. NT) >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
MOCK mean ± SD 53.6 ± 23.69 10.86 ± 4.66 21.61 ± 9.96 3.04 ± 1.64 0.83 ± 1.86 6.41 ± 3.36 0.93 ± 2.07
adj. p-value (vs. NT) 0.8859 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
DC mean ± SD 60.82 ± 36.16 17.19 ± 16.44 23.29 ± 14.19 2.09 ± 1.37 3.77 ± 4.37 4.02 ± 2.39 4.87 ± 5.22
adj. p-value (vs. >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
Mock)
RO mean ± SD 75.28 ± 13.56 16.86 ± 10.95 23.81 ± 16.74 2.56 ± 2.88 0.78 ± 0.83 3.89 ± 4.8 1.15 ± 1.28
adj. p-value (vs. 0.0204 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
Mock)
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis. Adjusted
(adj.) p values are included. Bold type: p < 0.05. % degranulated = (No. degranulated MC ÷ Total No. of
MC) x 100. MC, mast cells.
Table 22. Mast cell infiltration and degranulation at Day 21 (Exp-2, Timepoint-1).
Total No. MC/ No. Degranulated No. Perifollicular No. Follicular
% Degranulation % Perifollicular % Follicular
mm2 MC/ mm2 MC/ mm2 MC/ mm2
MOCK mean ± SD 28.06 ± 7.3 7.7 ± 5.33 25.28 ± 12.67 1.07 ± 1.03 0±0 3.32 ± 3.14 0±0
adj. p-value
AQ mean ± SD 53.69 ± 3.9 14.23 ± 4.47 26.62 ± 8.94 2.76 ± 1.29 0.66 ± 0.65 5.14 ± 2.41 1.26 ± 1.25
adj. p-value (vs. <0.0001 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
Mock)
MO mean ± SD 36.43 ± 4.47 12.16 ± 3.91 34.58 ± 14.4 2.56 ± 1.77 0.13 ± 0.2 7.31 ± 5.44 0.33 ± 0.47
adj. p-value (vs. 0.4883 >0.9999 0.3191 >0.9999 >0.9999 >0.9999 >0.9999
Mock)
WEN mean ± SD 43.84 ± 22.02 13.05 ± 10.29 30.4 ± 22.35 2.88 ± 1.27 0±0 7.27 ± 3.9 0±0
adj. p-value (vs. 0.0140 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
Mock)
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis.
Adjusted (adj.) p values are included. Bold type: p < 0.05. % degranulated = (No. degranulated MC
÷ Total No. of MC) x 100. MC, mast cells.
50
FDA-CU Final
Mock
Aquaphor
Monat
WEN
B. C.
Mock AQ MO WEN
degranulated mast cells/mm2
100
% degranulated mast cells
70
No. of infiltrating total or
60
80
* 50
60 ** 40
40 30
20
20
10
0 0
Total Degranulated Mock AQ MO WEN
51
FDA-CU Final
D. E.
No. of degranulated perifollicular Mock AQ MO WEN Mock AQ MO WEN
10 16
% degranulated perifollicular
and follicular mast cells/mm2
6 10
8
4 6
4
2
2
0 0
Perifollicular Follicular Perifollicular Follicular
Figure 15. Mast cell infiltration and degranulation at the anagen-telogen transition (Exp-2,
Timepoint-1). A. Representative images of toluidine blue-stained longitudinal sections of mouse dorsal
skin. Red arrows, mast cells (MCs). B. The average number of infiltrating and degranulating MCs per
mm2. C. % degranulation. D. Average number of degranulating perifollicular and follicular MCs. E. %
degranulation of perifollicular and follicular MCs. The graph represents the mean ± SD. n = 4-7 mice
per cohort. *, p < 0.05; **. p < 0.0001. Refer to Table 22 for the adjusted (adj.) p values for all data
points. AQ, Aquaphor; MO, Monat. Scale bar = 200 µm.
50 µm
Aquaphor
50 µm
Monat
50 µm
WEN
20 µm
52
FDA-CU Final
B. C.
100 Mock AQ MO WEN 70
degranulated mast cells/mm2 **
80 ** 60
** 50
60 40
40 * 30
20
20
10
0 0
Total Degranulated Mock AQ MO WEN
D. E.
Mock AQ MO WEN Mock AQ MO WEN
No. of degranulated perifollicular
% degranulated perifollicular
10 16
and follicular mast cells/mm2
Figure 16. Mast cell infiltration and degranulation at the telogen-anagen transition (Exp-2,
Timepoint-2). A. Representative images of toluidine blue-stained longitudinal sections of mouse dorsal
skin. Red arrows, red circles, mast cells (MCs). Red box, higher magnification of red circle. Black
arrows, regular MCs; magenta arrow, degranulating MCs. B. The average number of infiltrating and
degranulating MCs per mm2. C. % degranulation. D. Average number of degranulating perifollicular
and follicular MCs per mm2. E. % degranulation of perifollicular and follicular MCs. The graph
represents the mean ± SD. n = 4-7 mice per cohort. *, p < 0.05; **. p < 0.0001. Refer to Table 23 for the
adjusted (adj.) p values for all data points. AQ, Aquaphor; MO, Monat. Black scale bar = 100 µm.
Table 23. Mast cell infiltration and degranulation at Day 98 (Exp-2, Timepoint-2).
Total No. MC/ No. Degranulated No. Perifollicular
mm2 MC/ mm2 % Degranulation MC/ mm2 No. Follicular MC/ mm2 % Perifollicular % Follicular
Mock mean ± SD 36.58 ± 15.87 9.36 ± 7.24 23.36 ± 12.59 1.56 ± 0.52 0.09 ± 0.16 4.59 ± 1.89 0.25 ± 0.45
adj. p-value
AQ mean ± SD 40.6 ± 19.37 13.15 ± 10.36 27.9 ± 12.85 1.06 ± 1.2 0.32 ± 0.44 2.28 ± 2.39 0.54 ± 0.75
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
(vs. Mock)
MO mean ± SD 63.02 ± 1.82 21.55 ± 4.27 34.18 ± 6.55 0.96 ± 0.5 0.62 ± 0.59 1.51 ± 0.78 0.99 ± 0.92
adj. p-value <0.0001 0.0850 0.1748 >0.9999 >0.9999 >0.9999 >0.9999
(vs. Mock)
WEN mean ± SD 61.13 ± 17.76 26.58 ± 10.13 44.66 ± 14.95 1.51 ± 1.01 4.4 ± 3.83 2.42 ± 1.35 7.8 ± 6.34
adj. p-value <0.0001 0.0019 <0.0001 >0.9999 >0.9999 >0.9999 0.6478
(vs. Mock)
Two-way ANOVA with Bonferroni multiple comparison test was used for statistical analysis. Adjusted
(adj.) p values are included. Bold type: p < 0.05. % degranulated = (No. degranulated MC ÷ Total
No. of MC) x 100. MC, mast cells.
53
FDA-CU Final
Figure 17. Detection of mast cells by toluidine blue staining (A) and immunohistochemical
staining using mast cell tryptase antibody (B). Representative images are shown. n=5 tissue
sections per assay. Blue arrows, mast cells. Scale bar = 200 µm.
4.8.3.1 Evaluation of DevaCurl Low-Poo Delight Cleanser (Exp-1), and Aquaphor Baby Wash
& Shampoo, Monat Renew Shampoo, and WEN Sweet Almond Mint Cleansing
Conditioner (Exp-2)
Skin-resident macrophages have been implicated in the regulation of HF cycling, particularly during the
transition from the anagen phase to the catagen phase. Decreases in macrophages occur before the
onset of the anagen phase, and a selective reduction in the number of macrophages has been shown
to induce premature entry into the anagen phase [46]. Immunohistochemical staining of skin sections
using murine macrophage-specific anti-F4/80 antibody demonstrated higher numbers of F4/80+
macrophages in telogen skins (nontreated, CYP, DevaCurl, and Rogaine at D85 vs. D21, Exp-1)
whereas the number of F4/80+ macrophages in the mock was significantly decreased compared to the
nontreated cohort at Day 85 (Figs. 18 &19, Table 24). While these data corroborate those from prior
studies and suggest hair cycle-dependent fluctuations in macrophages, no significant changes were
detectable in the numbers of F4/80+ cells or in their distribution in mice treated with the test products
(Mock vs. DC, MO, WEN) (Figs. 18-21; Tables 24 & 25).
54
FDA-CU Final
4.8.3.2 Conclusion
All cohorts showed telogen-associated increases in the number of F4/80+ macrophages. No significant
differences were observed among the test products in the number and distribution of F4/80+
macrophages. Recent studies have shown that TREM2+ macrophages promote HF stem cell
quiescence during telogen and inhibit hair growth, suggesting the role of a distinct subset of dermal
macrophages in hair loss associated with prolonged or arrested telogen [47]. Further studies using
markers of specialized macrophages may help identify subpopulations relevant to alopecia.
Nontreated
CYP
Mock
DevaCurl
Rogaine
55
FDA-CU Final
Nontreated
CYP
Mock
DevaCurl
Rogaine
56
FDA-CU Final
Mock
Aquaphor
Monat
WEN
57
FDA-CU Final
Hair care products with high detergent properties can remove the outer cuticle, leave hair frizzy and
dull, and cause structural damage. To determine whether the test products cause structural
abnormalities of the hair shafts, dorsal hairs were evaluated for structural weaknesses, hair breakage,
focal bulge, and any abnormal features that represent defective hair [48]. In nontreated mice, the hair
shafts showed well-maintained structures with <2% of the hairs showing anomalies (Fig. 22, Table 26).
Pigmentation anomalies, including pigment clumping and depigmentation, were present in 10.00% of
hairs in the CYP cohort at Day 21 (p <0.0001) (Fig. 22, Table 26). While various defects in the hair
shafts were observed at Day 85 in all cohorts, the severity of the damages was not statistically
significant (Fig. 23, Table 27). Similarly, overall hair damage in Exp-1 was comparable to that in Exp-2
at both HF transitions (Figs. 24 & 25, Tables 28 & 29). Rather, structural weakness including hair
breakage was reduced in the WEN cohort at Day 98 (0.50% WEN vs. 4.50% mock, p=0.0112) (Fig. 25,
Table 29).
Figure 22. Hair shaft defects at the anagen-telogen transition (Exp-1, Timepoint-1). A.
Representative images of various hair shaft defects. Green arrow, focal bulge; blue arrow, pigment
clumping, depigmentation; red arrow, structural weakness, breakage. B. % damaged hairs. % damaged
hairs = (No. of damaged hairs ÷ Total No. of hairs) x 100. The graph represents the mean ± SD. n =7-8
mice per cohort (25-35 hair shafts were assessed per mouse.) *, p < 0.05. Refer to Table 26 for the
adjusted (adj.) p values for all data points. NT, nontreated; CYP, cyclophosphamide; DC, DevaCurl,
RO, Rogaine.
58
FDA-CU Final
Figure 23. Hair shaft defects at the telogen-anagen transition (Exp-1, Timepoint-2). A.
Representative images of various hair shaft defects. Green arrow, focal bulge; blue arrow, pigment
clumping, depigmentation; red arrow, structural weakness, breakage. B. % damaged hairs. % damaged
hairs = (No. of damaged hairs ÷ Total No. of hairs) x 100. The graph represents the mean ± SD. n =7-8
mice per cohort (25-35 hair shafts were assessed per mouse.) *, p < 0.05. Refer to Table 27 for the
adjusted (adj.) p values for all data points. NT, nontreated; CYP, cyclophosphamide; DC, DevaCurl,
RO, Rogaine.
59
FDA-CU Final
Figure 24. Hair shaft defects at the anagen-telogen transition (Exp-2, Timepoint-1). A.
Representative images of various hair shaft defects. Green arrow, focal bulge; blue arrow, pigment
clumping, depigmentation; red arrow, structural weakness, breakage. B. % damaged hairs. % damaged
hairs = (No. of damaged hairs ÷ Total No. of hairs) x 100. The graph represents the mean ± SD. n =7-8
mice per cohort (25-35 hair shafts were assessed per mouse.) *, p < 0.05. Refer to Table 28 for the
adjusted (adj.) p values for all data points. AQ, Aquaphor; MO, Monat.
60
FDA-CU Final
Figure 25. Hair shaft defects at the telogen-anagen transition (Exp-2, Timepoint-2). A.
Representative images of various hair shaft defects. Green arrow, focal bulge; blue arrow, pigment
clumping, depigmentation; red arrow, structural weakness, breakage. B. % damaged hairs. % damaged
hairs = (No. of damaged hairs ÷ Total No. of hairs) x 100. The graph represents the mean ± SD. n =7-8
mice per cohort (25-35 hair shafts were assessed per mouse.) *, p < 0.05. Refer to Table 29 for the p
values for all data points. AQ, Aquaphor; MO, Monat.
61
FDA-CU Final
For both Exp-1 and Exp-2, no substantial histopathological changes were observed in the liver tissues
collected at Timepoint 1 and Timepoint 2 in all cohorts. Fig. 26 shows the representative images of 7-8
mice per cohort.
62
FDA-CU Final
63
FDA-CU Final
In Exp-1, while no substantial differences were observed in average body weights in all cohorts, the
mock and DevaCurl cohorts showed less body weight gain compared to the nontreated mice (Table
30). In Exp-2, one mouse in the WEN cohort (Fig. 9D, red box) lost 20% of its body weight during the
second week of treatment. One mouse in the Aquaphor cohort died around Day 26. At Day 24, mice in
this cohort weighed between 16.8g and 20g, and the weight of this mouse was 18.7g. Therefore, the
cause of death is not likely due to weight loss. We also did not observe skin irritation, as well as any
other conditions interfering with eating or drinking in experimental animals. The total body weight gain
from Day 0 appeared to be higher in the mock and WEN cohorts. However, the average body weight at
Day 98 did not differ substantially in all cohorts (21.53g mock, 20.74g Aquaphor, 20.54g Monat, and
21.88g WEN) (Table 31).
64
FDA-CU Final
65
FDA-CU Final
66
FDA-CU Final
Anagen─telogen transition (TP-1): Compared to the mock cohort, mice treated with Aquaphor, Monat,
or WEN showed a higher proportion of HFs in the telogen phase at Day 21, suggesting accelerated
entry into the 1st telogen phase. However, this difference was not statistically significant.
Telogen─anagen transition (TP-2): Compared to the mock cohorts, a delay in progression to the 2nd
anagen phase was observed in mice treated with DevaCurl or WEN, with 46.39% of DevaCurl HFs and
71.2% of WEN HFs remaining in the 1st telogen phase at Day 85 and Day 98, respectively. The
observed delay in the WEN cohort was statistically significant.
Monat, WEN, and Rogaine were associated with increases in total mast cell numbers at the
telogen─anagen transition. In addition, WEN resulted in significant increases in mast cell activation.
Mast cell activity appeared to be preferentially localized within the HFs in WEN-treated mice. However,
this observation was not statistically significant and will require further investigation.
Overall, hair damage in the test cohorts was not statistically significantly higher than in the mock
cohorts.
67
FDA-CU Final
68
FDA-CU Final
Table 34. The presence of the test ingredients in the four selected products.
Name AQ DC MO WEN Function
1 Acetyl tetrapeptide‐3 ✓ Skin conditioning
2 Calendula Officinalis Extract ✓ ✓ Emollient, anti-inflammatory
3 Capixyl * ✓ Anti-inflammatory
4 Caryocar Brasiliense Fruit Oil (Pequi oil) ✓ Skin conditioning
5 Cinnamidopropyltrimonium chloride (CRODASORB UV‐ ✓ Antistatic
283), CATC
6 Citrus Limon (Lemon) Peel Oil ✓ Hair/skin conditioning,
fragrance
7 Cocamidopropyl Betaine (SurfProTM CAPB) ✓ ✓ ✓ Surfactant, viscosity control,
antistatic
8 Cocos Nucifera (Coconut) Oil ✓ Skin conditioning
9 Dextran 40 ✓** Viscosity control
10 Dextran 70 ✓** Viscosity control
11 Guar Hydroxypropyltrimonium Chloride ✓ ✓ Antistatic, viscosity control
12 Helianthus Annuus (Sunflower seed) Oil ✓ Emollient, fragrance,
antioxidant
13 Lavandula Angustifolia (Lavender) Oil fragrance
14 Methylchloroisothiazolinone (5‐Chloro‐2‐methyl‐4‐ ✓ Preservative
isothiazolin‐3‐one, MCI)
15 Methylisothiazolinone (MI) ✓ Preservative
16 Pisum Sativum (Pea) Peptide ✓ Skin conditioning
17 Polysorbate 60 Surfactant, emulsifier
18 Rosmarinus Officinalis Leaf Extract (Rosemary Oleoresin, ✓ ✓ Skin conditioning, anti-
ROE) microbial, fragrance
19 Solanum Lycopersicum (Tomato) Seed Oil ✓ Emollient, skin conditioning,
fragrance
20 Trifolium Pratense (Red Clover) Blossom Extract ✓ Skin conditioning
21 Vegetable oil (Olus oil) Emollient
AQ, Aquaphor Baby Wash & Shampoo; DC, DevaCurl Low-Poo Delight; MO, Monat Renew; WEN,
WEN Sweet Almond Mint Cleansing Conditioner
✓, Representing the presence of ingredients in test products.
*, Unable to obtain.
**, listed as Dextran on the product.
Experiments were carried out using two-dimensional cultures of (i) primary DPCs isolated from normal
human scalps (602-05A, Sigma-Aldrich) and (ii) NHEKs isolated from adult skin (00192627, Lonza).
Cells were maintained at 37 °C in 5% CO2 at 95% relative humidity. A serial dilution of the test
products/ingredients was prepared in tissue culture medium and filter-sterilized before use. Cell viability
69
FDA-CU Final
was assessed using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (G5430,
Promega), a quantitative assay that provides a readout of cell viability through the measurement of
metabolic activity.
Specifically, it measures the reducing potential of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] by metabolically active cells. In this assay
system, MTS is used in combination with an intermediate electron acceptor reagent (phenazine methyl
sulfate) to facilitate cell permeability. In healthy cells, MTS is converted by mitochondrial enzymes into
soluble purple formazan products. After two hours of incubation at 37°C, the number of viable cells was
determined by measuring absorbance at 490 nm in a microplate reader. The MTS assay was
performed following the manufacturer’s protocol. 1
Day 0: For each test product and ingredient, four 96-well plates (one plate each for four time points)
were seeded at 1,500 cells/well in 100 µl. The assay was performed over 6 days without replacing the
medium. To prevent evaporation and the risks associated with edge effects during the 6-day treatment,
we used 96-well plates with a built-in moat divided into four sectional reservoirs that can be filled with
sterile water (Thermo Scientific™ Nunc™ Edge 2.0). The number of cells seeded initially was optimized
to avoid confluent cultures, and no morphological changes were observed in nontreated control cells
during the treatment duration.
Day 1: Cells were treated with the test products/ingredients across a wide range of concentrations from
2.5E-06 to 10% (Table 35). The test concentrations for products/ingredients were determined based on
information from literature as well as the results, including solubility and cell viability, from the pilot
study (data not shown), to ensure the validity of the data. Five to six wells of cells were treated per
concentration (i.e., n=5-6 replicates per concentration). Each plate included nontreated cells, as well as
cells treated with minoxidil or cisplatin as controls. For test ingredients that have limited water solubility,
ethanol or dimethyl sulfoxide (DMSO) was used as solvents.
Days 2 – 7: MTS was added (20 µl/well) at 24, 48, and 72 hours (h), and 6 days (D) after treatment.
After two hours of incubation at 37°C in a humidified, 5% CO2 atmosphere, absorbance at 490 nm was
obtained using a microplate reader (Bio-Rad).
1
CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Technical Bulletin Part# TB169, Promega Corporation
70
FDA-CU Final
Each assay included cisplatin and minoxidil as controls. Cisplatin, an alkylating agent, has been shown
to inhibit proliferation and induce apoptosis in several hair follicle compartments, including dermal
papilla and matrix keratinocytes [53, 54]. Consistent with its cytotoxic effect on dividing cells, cisplatin
(100 µm) substantially reduced the viability of DPCs and NHEKs. Minoxidil is implicated in hair growth
and has been shown to shorten the telogen stage and prolong the anagen stage through both
proliferative and anti-apoptotic effects on the dermal papilla cells [23]. Overall increase in cell viability
was observed in DPCs and NHEKs treated with minoxidil (3 µm). Fig. 27 shows representative
datasets.
71
FDA-CU Final
Figure 27. Cytotoxicity assessment of minoxidil and cisplatin in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the assay controls for 24, 48, 72 hours (h), and 6 days (D) were assessed
by MTS assay. Cell viability (%) was obtained relative to nontreated control. Data in the tables are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. Highlighted values correspond to cell viability <
50%. *, p < 0.05; **, p < 0.0001.
72
FDA-CU Final
Statistical analysis was performed using GraphPad (Version 9.4.1.681, GraphPad Software, Inc.). Data
were analyzed using two-way ANOVA with Bonferroni multiple comparison test. Dose-response curve,
R2 (the coefficient of determination or goodness of fit), and IC50 (the half maximal inhibitory
concentrations) were obtained using GraphPad. Adjusted p values generated from Bonferroni tests,
IC50 values, and R2 values of >0.9 are included in the corresponding tables. Data are presented as
mean ± SD. p<0.05 was considered statistically significant. IC50 values that were unobtainable due to
no detectable level of cytotoxicity within the tested dose range and an R2 value of <0.9 were marked
with “N/A” (not available) in the tables.
5.3.4 Results
Test Products
Of the four hair care products (Table 32), we found Monat Renew shampoo to be the most cytotoxic to
DPCs, killing 80.94% of the cells in 24h at 0.04% (Fig. 28A, Table 36). On the other hand, WEN Sweet
Almond Mint cleansing conditioner had the least effect on DPC viability. WEN-induced cytotoxicity was
apparent only at 0.1% after 48h (Fig. 29A, Table 38), and the average IC50 of WEN was 7-fold higher
than that of MO (0.22% WEN vs. 0.03% MO) in DPCs. Similar cytotoxicity profiles were observed for
DevaCurl Low-Poo Delight Cleanser (Fig. 30A, Table 40) and Aquaphor Baby Wash & Shampoo (Fig.
31A, Table 42) in DPCs with an average IC50 of 0.05% for both. Although Aquaphor Baby Wash &
Shampoo appeared to be slightly more toxic than DevaCurl Low‐Poo Delight Cleanser at the early time
points, the difference was unsubstantial.
NHEKs were strikingly more sensitive to all the test products except for WEN. Although their effects
were less substantial at 24h, the IC50s of Monat Renew shampoo (Fig. 28B, Table 37), DevaCurl Low‐
Poo Delight Cleanser (Fig. 30B, Table 41), and Aquaphor Baby Wash & Shampoo (Fig. 31B, Table
43) were >25-fold lower than those observed in DPCs. NHEKs were less sensitive to WEN, with the
maximum decrease occurring at 0.1% 6 days after treatment (Fig. 29B, Table 39).
Figure 28. Cytotoxicity assessment of Monat Renew Shampoo (MO) in DPCs (A) and NHEKs (B).
Cells cultured in the presence of the test product for 24, 48, 72 hours (h), and 6 days (D) were
assessed by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs.
nontreated control). n=5-6 replicates per concentration.
73
FDA-CU Final
Table 36. Viability (%) of DPCs treated with Monat Renew Shampoo.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 4.47 89.14 ± 5.67 95.3 ± 2.77 98.1 ± 3.39 96.42 ± 4.23 103.25 ± 3.88 101.68 ± 5.02 19.06 ± 2.6 19.73 ± 4.74 21.19 ± 3.06 0.035 0.9787
adj. p-value 0.0001 0.4961 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 2.51 102.66 ± 3.99 100.84 ± 3.05 102.66 ± 4.17 101.4 ± 3.12 103.16 ± 6.16 91.44 ± 2.98 21.67 ± 13.97 14.17 ± 4.31 20.55 ± 6.47 0.031 0.9364
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 0.005 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 2.08 103.05 ± 4.47 100.61 ± 3.11 104.73 ± 3.36 105.95 ± 3.27 99.2 ± 4.46 81.01 ± 2.95 2.96 ± 0.33 5.73 ± 1.83 6.48 ± 0.97 0.025 0.9887
adj. p-value >0.9999 >0.9999 0.4812 0.139 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 5.25 96.36 ± 4.16 98.92 ± 1.88 102.18 ± 1.96 97.97 ± 3.36 86.08 ± 4.06 65.18 ± 3.82 0.96 ± 0.13 1.01 ± 0.23 1.03 ± 0.31 0.022 0.9854
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data
are represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values
correspond to cell viability< 50%.
Table 37. Viability (%) of NHEKs treated with Monat Renew Shampoo.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 3.97 95.5 ± 3.47 96.01 ± 4.66 92.53 ± 3.55 77.07 ± 4.96 54.55 ± 1.8 56.91 ± 1.85 60.39 ± 2.51 65.4 ± 4.05 71.44 ± 4.47 N/A N/A
adj. p-value 0.743 >0.9999 0.0379 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 2.44 88.71 ± 2.35 85.2 ± 4.09 71.65 ± 1.81 42.6 ± 1.23 41.2 ± 1.16 42.06 ± 1.94 43.69 ± 1.64 47.2 ± 2.59 49.22 ± 2.78 0.0010 0.9644
adj. p-value 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 15.74 91.23 ± 7.16 76.85 ± 8.96 64.99 ± 7.25 31.86 ± 1.14 32.49 ± 1.28 34.5 ± 1.76 37.08 ± 2.21 40.97 ± 2.21 45.21 ± 2.81 0.0008 0.9065
adj. p-value 0.0074 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 4.95 88.32 ± 5.29 65.79 ± 8.77 40.06 ± 4.48 12.89 ± 0.31 12.97 ± 0.3 13.71 ± 0.59 14.02 ± 0.65 14.89 ± 0.86 15.84 ± 1.2 0.0007 0.9828
adj. p-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Figure 29. Cytotoxicity assessment of WEN Sweet Almond Mint Cleansing Conditioner (WEN) in
DPCs (A) and NHEKs (B). Cells cultured in the presence of the test product for 24, 48, 72 hours (h),
and 6 days (D) were assessed by MTS assay. Dose response curves were obtained using GraphPad.
**, p < 0.0001 (vs. nontreated control). n=5-6 replicates per concentration.
74
FDA-CU Final
Table 38. Viability (%) of DPCs treated with WEN Sweet Almond Mint Cleansing Conditioner.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 4.58 99.27 ± 1.67 101.1 ± 2.54 99.27 ± 4.28 98.07 ± 3.71 98.62 ± 4.89 102.48 ± 3.35 102.93 ± 3.94 98.99 ± 3.42 58.74 ± 3.31 0.408 N/A
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001
48h mean ± SD 100 ± 4.47 98.52 ± 2.16 101.36 ± 1.9 96.11 ± 1.79 99.14 ± 2.03 99.14 ± 2.97 102.78 ± 3.3 104.69 ± 2.08 93.82 ± 2.35 24.89 ± 2.93 0.192 N/A
adj. p-value >0.9999 >0.9999 0.3645 >0.9999 >0.9999 >0.9999 0.1236 0.0115 <0.0001
72h mean ± SD 100 ± 2.78 96.62 ± 2.56 96.57 ± 2.19 101.37 ± 3.22 100.64 ± 3.17 99.85 ± 3.38 102.45 ± 3.37 106.51 ± 1.22 92.51 ± 5.18 11.94 ± 0.96 0.160 N/A
adj. p-value 0.6729 0.6363 >0.9999 >0.9999 >0.9999 >0.9999 0.0063 0.0009 <0.0001
6D mean ± SD 100 ± 2.86 99.57 ± 2 99.83 ± 2.28 96.36 ± 2.18 100.23 ± 1.82 96.76 ± 3.32 96.78 ± 1.63 97.18 ± 2.84 90.1 ± 2.59 2.05 ± 0.44 0.121 N/A
adj. p-value >0.9999 >0.9999 0.4948 >0.9999 0.7834 0.8084 >0.9999 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. Highlighted values correspond to cell viability<
50%. N/A, not available.
Table 39. Viability (%) of NHEKs treated with WEN Sweet Almond Mint Cleansing Conditioner.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 5.35 98.07 ± 7.29 102.03 ± 9.8 99.89 ± 5.8 99.46 ± 6.33 99.79 ± 5.21 100 ± 3.93 106.85 ± 4.73 111.78 ± 3.79 114.67 ± 4.33 N/A N/A
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 0.5219 0.0111 0.0006
48h mean ± SD 100 ± 6.77 91.93 ± 2.89 94.98 ± 10.32 90.75 ± 7.83 90.75 ± 7.6 86.44 ± 6.33 87.54 ± 6.54 91.14 ± 9.09 89.73 ± 8.53 89.73 ± 8.25 N/A N/A
adj. p-value 0.2321 >0.9999 0.0972 0.0972 0.0019 0.0058 0.1312 0.0426 0.0426
72h mean ± SD 100 ± 4.54 100.49 ± 4.64 102.5 ± 6.46 101.57 ± 6.8 101.32 ± 5.48 90.73 ± 7.69 89.55 ± 7.11 84.36 ± 4.66 79.7 ± 3.09 77.73 ± 4.56 N/A N/A
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 0.0956 0.0366 0.0002 <0.0001 <0.0001
6D mean ± SD 100 ± 4.31 98.98 ± 7.64 98.01 ± 6.91 98.36 ± 6.88 80.1 ± 5.37 74.23 ± 4.34 69.85 ± 4.74 61.68 ± 2.93 51.64 ± 4.41 50.22 ± 4.7 0.018 0.9252
adj. p-value >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Figure 30. Cytotoxicity assessment of DevaCurl Low-Poo Delight Cleanser (DC) in DPCs (A) and
NHEKs (B). Cells cultured in the presence of the test product for 24, 48, 72 hours (h), and 6 days (D)
were assessed by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001
(vs. nontreated control). n=5-6 replicates per concentration.
Table 40. Viability (%) of DPCs treated with DevaCurl Low-Poo Delight Cleanser.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 1.8 97.25 ± 3 95.31 ± 3.46 100.31 ± 2.93 98.47 ± 2.55 102.34 ± 3.39 99.9 ± 4.33 86.15 ± 3.79 52.13 ± 3.16 31.25 ± 3.03 0.079 0.9736
adj. p-value >0.9999 0.2563 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 1.71 89.34 ± 5.08 92.85 ± 12.54 99.36 ± 5.04 98 ± 3.02 101.72 ± 4.82 96.71 ± 4.27 70.09 ± 5.18 14.22 ± 1.69 7.35 ± 0.8 0.050 0.9651
adj. p-value <0.0001 0.0081 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 2.27 93.13 ± 1.65 95.6 ± 2.38 98.25 ± 2.7 100.13 ± 1.96 97.22 ± 1.62 87.06 ± 2.03 59.01 ± 2.39 9.05 ± 1.76 8.67 ± 1.49 0.043 0.9873
adj. p-value 0.0127 0.3566 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 4.2 98.01 ± 3.31 103 ± 4.05 104.82 ± 4.63 102.55 ± 3.78 94.88 ± 3.77 79.49 ± 2.33 50.65 ± 1.66 0.94 ± 0.12 1.07 ± 0.21 0.037 0.9803
adj. p-value >0.9999 >0.9999 0.2187 >0.9999 0.1508 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
75
FDA-CU Final
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values
correspond to cell viability< 50%.
Table 41. Viability (%) of NHEKs treated with DevaCurl Low-Poo Delight Cleanser.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 5.21 103.61 ± 5.7 109.74 ± 3.65 97.16 ± 5.38 90.81 ± 4.73 86.87 ± 3.42 83.37 ± 6.49 85.45 ± 5.86 61.71 ± 2.66 64.55 ± 4.04 N/A N/A
adj. p-value >0.9999 0.0878 >0.9999 0.1317 0.0048 0.0001 0.0012 <0.0001 <0.0001
48h mean ± SD 100 ± 10.33 96.26 ± 10.93 96.66 ± 11.79 81.45 ± 11.54 68.92 ± 8.9 63.55 ± 9.39 50.2 ± 6.81 48.41 ± 2.63 41.99 ± 1.66 41.33 ± 1.14 0.005 N/A
adj. p-value >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 9.25 95.95 ± 9.5 91.08 ± 7.94 78.01 ± 6.82 56.17 ± 7.15 45.44 ± 3.37 40.09 ± 7.49 36.62 ± 3.84 35.68 ± 2.02 37.45 ± 2.46 0.002 0.9441
adj. p-value >0.9999 0.1597 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 6.24 100.25 ± 4.71 99.48 ± 7.65 86.37 ± 7.26 39.35 ± 10.04 23.2 ± 2.57 13.45 ± 1.77 12 ± 0.26 11.77 ± 0.42 12.1 ± 0.38 0.003 0.9704
adj. p-value >0.9999 >0.9999 0.003 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values
correspond to cell viability< 50%. N/A, not available.
Figure 31. Cytotoxicity assessment of Aquaphor Baby Wash & Shampoo (AQ) in DPCs (A) and
NHEKs (B). Cells cultured in the presence of the test product for 24, 48, 72 hours (h), and 6 days (D)
were assessed by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001
(vs. nontreated control). n=5-6 replicates per concentration.
Table 42. Viability (%) of DPCs treated with Aquaphor Baby Wash & Shampoo.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 2.85 84.99 ± 5.7 92.86 ± 3.02 97.72 ± 5.05 97.83 ± 2.69 101.55 ± 3.75 104.86 ± 5.01 100.41 ± 2.76 19.17 ± 2.44 24.76 ± 6.4 0.067 0.9446
adj. p-value <0.0001 0.1649 >0.9999 >0.9999 >0.9999 0.9609 >0.9999 <0.0001 <0.0001
48h mean ± SD 100 ± 2.58 96.91 ± 3.33 94.29 ± 3.5 96.97 ± 5.91 101.31 ± 4.97 96.7 ± 4.06 96.84 ± 4.26 85.35 ± 4.6 8.68 ± 1.91 6.96 ± 2.91 0.054 0.9837
adj. p-value >0.9999 0.5286 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 1.22 94.99 ± 2.76 97.12 ± 2.56 98.27 ± 1.99 100.27 ± 2.81 98.67 ± 2.78 96.54 ± 2.51 80.38 ± 2.54 10.35 ± 0.62 11.15 ± 1.75 0.054 0.9913
adj. p-value 0.8676 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 2.55 84.45 ± 4.84 80.33 ± 24.64 96.05 ± 3.82 97.9 ± 4.05 96 ± 2.46 86.84 ± 3.78 69.2 ± 2.81 1.81 ± 0.28 1.76 ± 0.55 0.046 0.9799
adj. p-value <0.0001 <0.0001 >0.9999 >0.9999 >0.9999 0.0002 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%.
76
FDA-CU Final
Table 43. Viability (%) of NHEKs treated with Aquaphor Baby Wash & Shampoo.
0% 0.0001% 0.0005% 0.001% 0.005% 0.01% 0.02% 0.04% 0.08% 0.1% IC50 R2
24h mean ± SD 100 ± 6.72 94.68 ± 5.95 94.68 ± 6.48 92.4 ± 8.02 80.02 ± 7.31 71.01 ± 11.57 53.42 ± 0.82 54.51 ± 1.41 56.35 ± 1.78 57.98 ± 2.1 N/A N/A
adj. p-value 0.6035 0.6035 0.0827 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 5.65 88.8 ± 6.32 86.37 ± 6.85 79.07 ± 8.63 60.5 ± 6.59 34.7 ± 0.55 33.73 ± 0.72 34.01 ± 0.69 34.42 ± 0.35 35.54 ± 0.55 0.003 0.9396
adj. p-value 0.0013 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 5.05 91.9 ± 8.58 88.59 ± 10.72 78.36 ± 8.05 46.59 ± 3.44 26.24 ± 0.72 26.28 ± 0.85 26.58 ± 0.85 27.47 ± 0.83 29.55 ± 1.64 0.002 0.9526
adj. p-value 0.0498 0.001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 1.28 95.77 ± 5.9 78.79 ± 4.64 63.24 ± 4.43 15.67 ± 2.01 12.46 ± 0.45 12.61 ± 0.35 13.18 ± 0.35 14.3 ± 0.56 15.52 ± 0.78 0.001 0.9755
adj. p-value >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%.
Test Ingredients
Acetyl tetrapeptide-3
Acetyl tetrapeptide-3 is a four amino acid peptide, typically used at 0.5–5% in cosmetics. Herbal extract
combinations containing acetyl tetrapeptide-3 have been implicated in hair growth [55, 56], and a
mixture of acetyl tetrapeptide-3 (326 ppm, 0.0326%) and red clover extract is marketed as Follicle
Booster (MakingCosmetics). The effects of acetyl tetrapeptide-3 alone on hair growth are unknown. In
DPCs, acetyl tetrapeptide-3 resulted in an overall 60% decrease in cell viability, but only in cells
exposed to the highest concentration (1%) for 6 days (Fig. 32A, Table 44). The overall cytotoxicity
profiles of acetyl tetrapeptide-3 in NHEKs were similar to those observed in DPCs. However, NHEKs
were somewhat more sensitive at later time points. At day 6, the viability of NHEKs treated with 0.5%
acetyl tetrapeptide-3 was reduced to 25.3% (vs. 64.3% in DPCs) (Fig. 32B, Table 45).
Figure 32. Cytotoxicity assessment of Acetyl tetrapeptide-3 in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed
by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated
control). n=5-6 replicates per concentration.
77
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
In contrast, calendula extract increased NHEK viability at 24h, followed by gradual decreases at later
time points (Fig. 33B, Table 47). At concentrations <0.05%, calendula extract appeared to promote cell
growth, while concentrations >0.2% were cytotoxic, leading to a reduction in cell viability of >50%.
However, this effect was apparent only at day 6. Although uncertain, this decrease at later time points
could be due to nutrition deprivation and/or pH changes in the medium.
78
FDA-CU Final
Figure 33. Cytotoxicity assessment of Calendula extract in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed
by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated
control). n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
79
FDA-CU Final
showed significant dose-dependent increases in cell viability, with the maximum increase occurring 24h
after treatment (Fig. 34A, Table 48). In NHEKs, substantial increases in cell viability were observed at
concentrations >0.125%, which peaked at 24h and then gradually decreased at later time points. The
maximum increase in cell viability, 111.18%, was observed at 24h in cells treated with 0.25% (Fig. 34B,
Table 49).
Figure 34. Cytotoxicity assessment of Pequi oil in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
80
FDA-CU Final
CATC is not available, although similar quaternary ammonium compounds have been shown to cause
reproductive toxicity in mice [63]. A typical concentration range of quaternary ammonium compounds in
cosmetic products is between 150 – 400 ppm (0.015 – 0.04%). At concentrations > 0.5%, we observed
a clear dose- and time-dependent cytotoxicity in DPCs (Fig. 35A, Table 50). DPCs treated with 0.5%
CATC showed a 75.26% reduction in cell viability 48 hours after treatment, which was further reduced
to 2.42% at day 6 (Table 50). Concentrations > 2% were toxic, killing most cells in 24 hours. In stark
contrast to the acute cytotoxicity observed in DPCs, CATC caused only modest decreases in NHEK
viability. For example, at 24h, >60% of NHEKs remained viable when exposed to >2.5% of CATC (Fig.
35B, Table 51).
Figure 35. Cytotoxicity assessment of CATC in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
81
FDA-CU Final
Figure 36. Cytotoxicity assessment of Lemon peel oil in DPCs (A) and NHEKs (B). Cells cultured
in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Table 52. Viability (%) of DPCs treated with Lemon peel oil.
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 3.74 106.02 ± 4.1 103.05 ± 2.12 110.73 ± 3.39 126.25 ± 3.63 157.41 ± 3.6 182.2 ± 2.16 N/A 0.9862
adj. p-value 0.0302 0.9098 <0.0001 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 3.17 108.1 ± 2.63 103.85 ± 3.38 118.23 ± 3.7 140.65 ± 5.06 158.88 ± 5.05 171.82 ± 4.04 N/A 0.9728
adj. p-value 0.0011 0.4228 <0.0001 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 3.09 113.82 ± 3.72 96.53 ± 3.14 113.09 ± 2.45 143.95 ± 4.5 145.25 ± 5.16 146.94 ± 2.67 N/A 0.9171
adj. p-value <0.0001 0.6148 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 3.39 103.46 ± 3.19 101.33 ± 3.05 113.44 ± 2.55 144.88 ± 2.88 115.5 ± 5.23 91.18 ± 4.68 N/A N/A
adj. p-value 0.6212 >0.9999 <0.0001 <0.0001 <0.0001 0.0003
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Table 53. Viability (%) of NHEKs treated with Lemon peel oil.
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 3.84 104.56 ± 3.2 106.08 ± 5.11 106.08 ± 1.9 110.88 ± 3.89 131.11 ± 2.24 139.77 ± 4.79 N/A 0.9277
adj. p-value >0.9999 >0.9999 >0.9999 0.086 <0.0001 <0.0001
48h mean ± SD 100 ± 4.7 102.92 ± 5.11 105.36 ± 4.9 106.62 ± 3.37 121.21 ± 3.42 139.51 ± 4.78 146.06 ± 7.36 N/A 0.9294
adj. p-value >0.9999 >0.9999 0.7993 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 5.27 113.47 ± 10.93 115.02 ± 9.79 132.39 ± 8.82 159.6 ± 7.19 160.81 ± 11.02 172.71 ± 12.89 N/A N/A
adj. p-value 0.0154 0.0048 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 4.31 100.82 ± 1.81 100.7 ± 4.29 105.86 ± 3.87 108.2 ± 8.25 86.47 ± 19.71 69.78 ± 13.2 N/A N/A
adj. p-value >0.9999 >0.9999 >0.9999 0.3811 0.0148 <0.0001
82
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Figure 37. Cytotoxicity assessment of CAPB in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
83
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Figure 38. Cytotoxicity assessment of Coconut oil in DPCs (A) and NHEKs (B). Cells cultured in
the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
84
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Figure 39. Cytotoxicity assessment of Dextran 40 in DPCs (A) and NHEKs (B). Cells cultured in
the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
85
FDA-CU Final
Figure 40. Cytotoxicity assessment of Dextran 70 in DPCs (A) and NHEKs (B). Cells cultured in
the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
86
FDA-CU Final
substantial cytotoxicity observed in DPCs, Guar increased NHEK viability at 24h, followed by gradual
decreases at later time points (Fig. 41B, Table 63).
Figure 41. Cytotoxicity assessment of Guar in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
87
FDA-CU Final
Figure 42. Cytotoxicity assessment of Sunflower seed oil in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed
by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated
control). n=5-6 replicates per concentration.
Table 64. Viability (%) of DPCs treated with Sunflower seed oil
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 3.82 94.43 ± 2.79 98.73 ± 3.62 99.72 ± 3.73 113.12 ± 3.7 147.89 ± 3.77 196 ± 5.76 N/A 0.9857
adj. p-value 0.0263 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 2.21 103 ± 2.98 100.5 ± 3.11 100.65 ± 1.78 109.19 ± 4.36 133.35 ± 3.03 164.6 ± 3.88 N/A 0.982
adj. p-value 0.7304 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 4.47 99.46 ± 2.4 95.45 ± 2.97 100.95 ± 2.59 106.17 ± 2.83 127.73 ± 2.78 154.56 ± 3.74 N/A 0.9735
adj. p-value >0.9999 0.1159 >0.9999 0.01 <0.0001 <0.0001
6D mean ± SD 100 ± 3.65 100.82 ± 3.13 98.56 ± 2.42 101.94 ± 1.65 102.69 ± 2.51 113.83 ± 2.53 125.74 ± 3.9 N/A 0.9146
adj. p-value >0.9999 >0.9999 >0.9999 0.9896 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Table 65. Viability (%) of NHEKs treated with Sunflower seed oil.
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 6 108.44 ± 4.62 106.44 ± 3.04 112.15 ± 4.08 116.72 ± 6.87 126.23 ± 5.91 141.49 ± 3.89 N/A N/A
adj. p-value 0.4836 >0.9999 0.1092 0.004 <0.0001 <0.0001
48h mean ± SD 100 ± 4.2 110.89 ± 8.27 119.69 ± 10.81 122.16 ± 6.93 122.63 ± 11.29 129.42 ± 4.81 144.71 ± 15.18 N/A N/A
adj. p-value 0.0844 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 4.04 113.52 ± 7.23 117.78 ± 8.69 119.71 ± 10.62 128.16 ± 8.74 131.05 ± 10.11 143.85 ± 13.91 N/A N/A
adj. p-value 0.0333 0.0018 0.0004 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 5.23 103.13 ± 3.74 102.91 ± 4.11 108.2 ± 1.55 103.63 ± 6.05 108.57 ± 3.11 104.57 ± 6.46 N/A N/A
adj. p-value >0.9999 >0.9999 0.3786 >0.9999 0.3125 >0.9999
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. N/A, not available.
88
FDA-CU Final
Figure 43. Cytotoxicity assessment of Lavender oil in DPCs (A) and NHEKs (B). Cells cultured in
the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
89
FDA-CU Final
MCI/MI, marketed as Kathon® CG) [71], the use of MCI/MI has been limited to 7.5 ppm in the US.
However, because MI is assumed to be less allergenic than MCI, MI as a stand-alone preservative is
permitted at higher concentrations (up to 100 ppm, 0.01%) [72]. In our study, the cytotoxicity of MCI
and MI was assessed separately with concentrations ranging from 0.00001% to 0.1%. MCI was
extremely toxic to DPCs, killing >90% of the cells in 24h at > 0.00005% (Fig. 44A, Table 68). MI was
also toxic; however, the average IC50 of MCI was 10- to15-fold lower than that of MI (1.8E-05 vs. 2.3E-
04%) in DPCs and longer exposure was required for MI to achieve a reduction of cell viability >90%
(Fig. 45A,Table 70). In contrast to the acute cytotoxicity observed in DPCs, >60% of NHEKs remained
viable at 24h when exposed to >0.005% of MCI (Fig. 44B,Table 69) and MI caused no substantial
reduction in NHEK viability within 48h (Fig. 45B,Table 71).
Figure 44. Cytotoxicity assessment of MCI in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
90
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Figure 45. Cytotoxicity assessment of MI in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
91
FDA-CU Final
Figure 46. Cytotoxicity assessment of Pea extract in DPCs (A) and NHEKs (B). Cells cultured in
the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
92
FDA-CU Final
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Polysorbate 60
Polysorbate 60 is obtained by esterification of sorbitol with one or three molecules of a fatty acid
including stearic, lauric, oleic, and palmitic acid. It is a nonionic, multi-purpose emulsifying agent and
also functions as a thickener. The typical use level is 1–10% in cosmetic products; however, testing
concentrations >2.5% was not feasible due to insolubility. Although decreases in cell viability were
observed at concentrations >0.5%, its effect was not apparent at later time points (72h and 6D) (Fig.
47A, Table 74). In contrast, polysorbate 60 at concentrations >0.01% was cytotoxic in NHEKs and
reduced cell viability to 22.85% at day 6 (Fig. 47B, Table 75).
Figure 47. Cytotoxicity assessment of Polysorbate 60 in DPCs (A) and NHEKs (B). Cells cultured
in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
93
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Figure 48. Cytotoxicity assessment of Rosemary leaf extract in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed
by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated
control). n=5-6 replicates per concentration.
94
FDA-CU Final
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
Figure 49. Cytotoxicity assessment of Tomato seed oil in DPCs (A) and NHEKs (B). Cells cultured
in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS
assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control).
n=5-6 replicates per concentration.
Table 78. Viability (%) of DPCs treated with Tomato seed oil.
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 6.16 96.39 ± 3.77 95.65 ± 6.09 101.62 ± 2.66 119.08 ± 5.09 144.86 ± 5.77 170.19 ± 12.38 N/A 0.9393
adj. p-value 0.7648 0.6053 0.9929 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 2.41 101.44 ± 4.09 100.94 ± 4.26 105.45 ± 2.19 126.44 ± 2.44 140.41 ± 6.89 159.57 ± 10.21 N/A 0.9274
adj. p-value 0.9955 0.9996 0.377 <0.0001 <0.0001 <0.0001
95
FDA-CU Final
Table 79. Viability (%) of NHEKs treated with Tomato seed oil.
0% 0.0001% 0.001% 0.01% 0.1% 0.5% 1% IC50 R2
24h mean ± SD 100 ± 14.56 88.83 ± 7.62 90.86 ± 2.31 88.96 ± 6.96 90.61 ± 10.89 94.54 ± 5.65 106.73 ± 14.25 N/A N/A
adj. p-value 0.2476 0.565 0.2616 0.5127 >0.9999 >0.9999
48h mean ± SD 100 ± 5.33 93.95 ± 7.69 94.22 ± 5.68 94.94 ± 8.23 93.58 ± 6.32 102.89 ± 10.8 116.17 ± 10.86 N/A N/A
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 0.0202
72h mean ± SD 100 ± 8.49 93.64 ± 11.44 95.82 ± 10.56 91.55 ± 11.39 97.59 ± 12.73 102.9 ± 7.24 102.41 ± 8.52 N/A N/A
adj. p-value >0.9999 >0.9999 0.7282 >0.9999 >0.9999 >0.9999
6D mean ± SD 100 ± 4.78 101.12 ± 7.49 101.4 ± 11.4 99.86 ± 9.82 97.44 ± 9.53 105.13 ± 12.03 100 ± 7.59 N/A N/A
adj. p-value >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. N/A, not available.
Figure 50. Cytotoxicity assessment of Red clover extract in DPCs (A) and NHEKs (B). Cells
cultured in the presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed
96
FDA-CU Final
by MTS assay. Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated
control). n=5-6 replicates per concentration.
Table 80. Viability (%) of DPCs treated with Red clover flower extract.
0% 0.001% 0.01% 0.1% 1% 2% 5% 8% 10% IC50 R2
24h mean ± SD 100 ± 4.09 97.52 ± 2.74 99.02 ± 2.13 98.63 ± 3.86 95.05 ± 2.03 100.85 ± 2.85 88.27 ± 3.04 71 ± 2.54 67.48 ± 3.46 14.090 0.9114
adj. p-value >0.9999 >0.9999 >0.9999 0.0292 >0.9999 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 3.73 97.67 ± 4.28 97.06 ± 2.29 94.66 ± 2.67 94.54 ± 3.13 92.78 ± 2.45 73.35 ± 2.32 54.8 ± 1.96 50.64 ± 2.97 9.751 0.96
adj. p-value >0.9999 0.6566 0.0139 0.0111 0.0002 <0.0001 <0.0001 <0.0001
72h mean ± SD 100 ± 3.86 100.1 ± 2.01 99.77 ± 2.08 99.11 ± 3.87 89.03 ± 3.13 81.85 ± 3.21 53.78 ± 4.83 33.82 ± 1.68 26.96 ± 4.88 5.259 0.9852
adj. p-value >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 2.66 100.58 ± 3.02 99.42 ± 0.9 97.84 ± 2.44 86.29 ± 1.48 75.51 ± 1.82 47.28 ± 2.2 22.22 ± 0.87 15.79 ± 0.96 4.041 0.9894
adj. p-value >0.9999 >0.9999 >0.9999 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%.
Table 81. Viability (%) of NHEKs treated with Red clover flower extract.
0% 0.001% 0.01% 0.1% 1% 2% 5% 8% 10% IC50 R2
24h mean ± SD 100 ± 9.22 80.8 ± 9.36 77.46 ± 6.96 81.55 ± 4.24 93.81 ± 9.51 107.55 ± 6.83 132.45 ± 6.98 140.87 ± 5.98 147.06 ± 8.97 N/A N/A
adj. p-value <0.0001 <0.0001 <0.0001 0.6674 0.2804 <0.0001 <0.0001 <0.0001
48h mean ± SD 100 ± 5.87 81.62 ± 7.47 90.39 ± 8.71 96 ± 5.03 99.91 ± 3.52 101.11 ± 3.04 87.07 ± 3.99 89.79 ± 4.3 89.53 ± 5.1 N/A N/A
adj. p-value <0.0001 0.0602 >0.9999 >0.9999 >0.9999 0.0029 0.0367 0.0295
72h mean ± SD 100 ± 7.05 90.89 ± 8.82 91.25 ± 4.82 96.86 ± 10.72 98.4 ± 9.86 92.06 ± 4.51 57.26 ± 5.39 58.74 ± 4.08 60.89 ± 5.06 N/A N/A
adj. p-value 0.0897 0.1191 >0.9999 >0.9999 0.214 <0.0001 <0.0001 <0.0001
6D mean ± SD 100 ± 2.72 101.14 ± 3.66 99.74 ± 5.45 98.93 ± 5.1 87.17 ± 3.69 62.9 ± 2.17 13.04 ± 0.26 13.89 ± 0.37 17.06 ± 1.43 2.141 0.9895
adj. p-value >0.9999 >0.9999 >0.9999 0.0032 <0.0001 <0.0001 <0.0001 <0.0001
Cell viability (% relative to nontreated control), IC50, and R2 were obtained using GraphPad. Data are
represented as the mean ± SD; n=5-6 replicates per concentration. Adjusted (adj.) p values were
generated using Bonferroni multiple comparison test. R2 > 0.9 is indicated. Highlighted values correspond
to cell viability< 50%. N/A, not available.
97
FDA-CU Final
Figure 51. Cytotoxicity assessment of Olus oil in DPCs (A) and NHEKs (B). Cells cultured in the
presence of the test ingredient for 24, 48, 72 hours (h), and 6 days (D) were assessed by MTS assay.
Dose response curves were obtained using GraphPad. **, p < 0.0001 (vs. nontreated control). n=5-6
replicates per concentration.
• All test products demonstrated significant cytotoxicity within 24h in DPCs; WEN product showed
cytotoxicity only at the highest concentration (0.1%) at 24 h.
• In NHEKs, the cytotoxic effects of Monat, Aquaphor, and DevaCurl were apparent from 48h.
• Compared to the other test products, Monat was the most cytotoxic in both DPCs and NHEKs.
WEN was the least cytotoxic in DPCs and non-cytotoxic in NHEKs.
• Of the 20 test ingredients, 11 test ingredients demonstrated varying degrees of cytotoxicity in
DPCs within 72h. Of these, seven induced acute cytotoxicity in DPCs, decreasing cell viability by
more than 50% within 24h. These included MCI, MI, CAPB, Lavender oil, Polysorbate 60, CATC,
and Pea extract.
• Of all ingredients tested, MCI and MI were the most cytotoxic, with IC50 values of 2.13E-05% and
0.0003%, respectively, at 24h, followed by Rosemary extract, which had an IC50 value of 0.001%
at 48h.
• Coconut oil, Dextrans 40 and 70, Lemon peel oil, Pequi oil, Sunflower seed oil, Tomato seed oil,
and Vegetable oil were noncytotoxic and produced overall increases in cell viability in both DPCs
and NHEKs.
98
FDA-CU Final
2. Test Ingredients
Cytotoxicity Status*
DPCs: DPCs: NEHKs: NEHKs:
Test Ingredient IC50 (%) Test Ingredient IC50 (%)
MCI 2.13E-05
MI 0.0003
CAPB 0.067
Cytotoxic at 24h Lavender oil 0.097
Polysorbate 60 0.322
CATC 0.573
Pea extract 1.162
MCI 2.60E-04
Cytotoxic at 48h Rosemary extract 0.001
CATC 0.134
*, Cytotoxicity was defined as < 50% cell viability at a given experimental condition.
N/A, Not available.
99
FDA-CU Final
We evaluated three in vitro assay platforms in DPCs: 1) the Click-iT TUNEL assay (Thermo Fisher), a
modified TUNEL assay that incorporates an alkyne-modified dUTP at the 3'-OH ends of fragmented
DNA and enables microscope imaging of apoptotic cells; 2) the Caspase-Glo 3/7 assay system
(Promega) which detects Caspase 3/7 activity using a proluminescent DEVD-aminoluciferin substrate;
and 3) the RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega) which measures the
real-time PS exposure on the outer leaflets of cell membranes during the apoptotic process.
The TUNEL assay involves imaging analysis; hence, the quantitation of apoptotic cells was
cumbersome and not robust. Both the Caspase 3/7 and Annexin V Apoptosis and Necrosis assays
detect luminescence-based apoptosis; however, the caspase 3/7 assay was less sensitive than the
annexin V and necrosis assay, and further optimization was needed. For these reasons, we chose the
annexin V apoptosis and necrosis assay, which enables continuous monitoring of apoptosis and
fluorescence-based detection of necrosis that depends on the loss of cell membrane integrity.
The assay used in this study allows the measurements of luminescent signals generated by apoptotic
cells and fluorescent signals generated by necrotic cells.
Phospholipids of the cell membrane are asymmetrically distributed on the inner and outer leaflets of the
lipid bilayers. PS is located on the inner leaflets, and other phospholipids (e.g., phosphatidylcholine,
sphingomyelin) are located on the external leaflets. During apoptosis, PS externalizes to the outside
surfaces of cell membranes. The cell membrane is still intact during this process. Because PS has a
high affinity for the anticoagulant protein annexin V, the binding of PS to various annexin V conjugates
is commonly used to detect apoptosis.
100
FDA-CU Final
The luminescence-based apoptosis assay utilized two annexin fusion V proteins containing
complementary subunits of luciferase. In cells undergoing apoptosis, annexin V–luciferase conjugates
bind to the externalized PS, producing luminescent signals (Table 85).
The fluorescence-based necrosis assay measured exposed DNA that occurs because of loss of cell
membrane integrity and subsequent cell lysis. The assay system uses a cell-impermeable,
profluorescent DNA dye that is excluded from viable cells but preferentially stains the dead cells’ DNA.
When the dye binds to DNA, the dye’s fluorescent properties are substantially enhanced. Therefore, the
fluorescent signal produced by the dye binding to the dead cells’ DNA is proportional to the necrosis.
In vitro, cultured cells that are undergoing apoptosis eventually lose membrane integrity and release
their cytoplasmic contents into the culture medium (post-apoptotic necrosis). In post-apoptotic necrosis,
luminescence is preceded by temporal increases in fluorescence. The concurrent increases in
luminescence and fluorescence indicate necrotic cell death, in which the luminescent signal is
generated by the non-specific binding of annexin V to PS present in cell debris. In general, the
fluorescence signal indicates necrosis.
The test ingredients associated with decreases in DPC viability of > 50% within 72h were assessed for
apoptosis induction in DPCs (Table 86).
101
FDA-CU Final
Treatment
No. of
Test Ingredients* Vehicle C1 (%) C2 (%) C3 (%) Duration
treatments
(hours)
20%
9 Polysorbate 60 0.16 0.32 0.64 1 15, 24, 48, 72
Ethanol**
Rosmarinus Officinalis Leaf Extract
10 2.5% DMSO 0.0005 0.001 0.002 1 48, 72
(Rosemary Oleoresin, ROE)
Trifolium Pratense (Red Clover)
11 Medium 2.65 5.3 10.6 1 15, 24, 48, 72
Blossom Extract
2. Assay Controls
Treatment
No. of
Assay Controls Vehicle Concentration Duration
treatments
(hours)
12 Minoxidil 100% Ethanol** 3 µm 1 24, 48, 72
13 Cisplatin DMSO 100 µm 1 24, 48, 72
*, Annexin V binding assay was done only for ingredients shown to inhibit DPC viability within 72h by
MTS assay.
**, Refer to Table 4 for the preparation of the stock solutions.
Experiments were performed using the annexin V apoptosis and necrosis assay (JA1011, Promega)
following the manufacturer's protocol. 2 Each test ingredient was tested at three concentrations in
DPCs: one that was two-fold lower than the IC50 (C1); IC50 (C2), determined based on the MTS
results; and a concentration that was two-fold higher than the IC50 (C3) (Table 86). The assay included
nontreated DPCs (C0) and those treated with cisplatin (100 µm) or minoxidil (3 µm) as controls. The
luminescence (resulting from annexin V binding to PS) and fluorescence (resulting from a cell-
impermeant DNA dye binding to dead cells’ DNA) were measured using a multimode plate reader
(Tecan infinite 200Pro). Four replicates (n=4) were tested per concentration per timepoint. The
experiments were performed twice.
Statistical analysis was performed using two-way analysis of variance (ANOVA) with Bonferroni
multiple comparison test (GraphPad, version 9.4.1.681, GraphPad Software, Inc.). Data are presented
as fold changes in luminescence (indicating annexin V binding/apoptosis) or in fluorescence (indicating
DNA staining/necrosis) relative to the nontreated control (mean ± SD; n=4 replicates per concentration
per timepoint). Adjusted p values generated from Bonferroni tests are included in the tables. p<0.05
was considered statistically significant. The luminescence and fluorescence signal intensities that are
lower than those of the nontreated controls (C0) or that are detectable only at the background level
suggest no activity at a given concentration and time point. Such values are indicated as “ND” (not
detectable) in the tables.
DPCs treated with cisplatin showed time-dependent increases in annexin V binding, while the
fluorescence resulting from the DNA staining of necrotic cells remained at a level comparable to that of
the nontreated control. Consistent with the MTS results, cisplatin-induced apoptosis was detectable in
cells treated for > 48h and showed 2- and 9-fold increases (vs. the nontreated control) at 48h and 72h,
respectively (Table 87). In line with the growth-promoting effects of minoxidil in DPCs, minoxidil-treated
DPCs showed neither detectable annexin V binding nor necrosis (Table 88). These data validate the
2
RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay, Technical Manual # TM507, Promega Corporation
102
FDA-CU Final
utility of this assay in assessing apoptosis in DPCs. No luminescent and fluorescent signals were
detectable at 24h in DPCs treated with cisplatin. Therefore, apoptosis and necrosis were assessed at
48h and 72h.
5.4.7 Results
Rosmarinus Officinalis (Rosemary) Leaf Extract, Lavandula Angustifolia (Lavender) Oil, and
Guar Hydroxypropyltrimonium Chloride (Guar)
Rosemary extract resulted in dose- and time-dependent increases in annexin V-positive/necrosis-
negative cells (Table 89). Compared to the nontreated cells (C0), an IC50 dose (C2) of rosemary
extract caused a 2.16-fold (p<0.0001) increase in annexin V binding at 48h and a 4.89-fold (p<0.0001)
increase at 72h. The highest concentration tested (C3, 0.002%) showed the most apoptotic activity,
with a 6.49-fold increase (p<0.0001, 72h) (Table 89). The IC50 doses of lavender oil and Guar also
induced 2.4-fold (p<0.0001) and 3.4-fold (p<0.0001) increases in annexin V binding, respectively, at
72h (Tables 90 & 91). The absence of necrosis signals indicated that apoptosis was likely the primary
mechanism of cells death induced by these ingredients.
103
FDA-CU Final
104
FDA-CU Final
105
FDA-CU Final
Table 100. Red Clover, Polysorbate 60, MCI, MI, and CAPB at 15h.
Apoptosis Apoptosis Apoptosis Apoptosis Necrosis Necrosis Necrosis Necrosis
C0 C1 C2 C3 C0 C1 C2 C3
Red Clover mean ± SD 1 ± 0.03 ND ND ND 1 ± 0.02 1.34 ± 0.04 1.87 ± 0.03 2.67 ± 0.02
adj. p-value <0.0001 <0.0001 <0.0001
Polysorbate mean ± SD 1 ± 0.02 ND ND ND 1 ± 0.02 ND ND 1.14 ± 0.01
60
adj. p-value <0.0001
MCI mean ± SD 1 ± 0.03 ND ND ND 1 ± 0.01 ND ND ND
adj. p-value
MI mean ± SD 1 ± 0.03 1.02 ± 0.02 1.13 ± 0.02 ND 1 ± 0.01 1.14 ± 0 1.31 ± 0.02 1.25 ± 0.02
adj. p-value 0.5459 <0.0001 <0.0001 <0.0001 <0.0001
CAPB mean ± SD 1 ± 0.03 ND ND ND 1 ± 0.01 ND 1.02 ± 0.01 1.04 ± 0.01
adj. p-value 0.4681 0.0181
Data are represented as the mean ± SD; n=4. Adjusted (adj.) p values were generated using
Bonferroni multiple comparisons test. Highlighted values correspond to a fold change > 2. ND, not
detectable.
106
FDA-CU Final
Table 101. Red Clover, Polysorbate 60, MCI, MI, and CAPB at 24h.
Apoptosis Apoptosis Apoptosis Apoptosis Necrosis Necrosis Necrosis Necrosis
C0 C1 C2 C3 C0 C1 C2 C3
Red Clover mean ± SD 1 ± 0.03 ND ND ND 1 ± 0.01 1.26 ± 0.04 1.73 ± 0.02 2.44 ± 0.01
adj. p-value <0.0001 <0.0001 <0.0001
Polysorbate mean ± SD 1 ± 0.02 ND ND ND 1 ± 0.02 ND ND 1.07 ± 0.02
60
adj. p-value <0.0001
MCI mean ± SD 1 ± 0.03 ND 1.05 ± 0.02 1.04 ± 0.01 1 ± 0.01 ND ND ND
adj. p-value 0.0012 0.0174
MI mean ± SD 1 ± 0.03 1.05 ± 0.01 1.19 ± 0.02 1.15 ± 0.04 1 ± 0.01 1.31 ± 0.01 1.64 ± 0.04 1.48 ± 0.02
adj. p-value 0.0013 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
CAPB mean ± SD 1 ± 0.03 ND ND ND 1 ± 0.01 ND 1.03 ± 0.01 1.04 ± 0.01
adj. p-value 0.1598 0.0212
Data are represented as the mean ± SD; n=4. Adjusted (adj.) p values were generated using
Bonferroni multiple comparisons test. Highlighted values correspond to a fold change > 2. ND, not
detectable.
5.4.8 Summary
Table 102 summarizes the results of the apoptosis and necrosis assessment.
• Three ingredients (i.e., rosemary extract, guar, lavender oil) induced apoptosis at IC50 doses.
• Rosemary extract showed a time-dependent increase in annexin V binding (2.16-fold at 24h, 4.89-
fold at 48h).
• Guar and lavender oil-induced apoptosis was detectable at 48h.
107
FDA-CU Final
• CATC and pea extract caused robust increases in DNA dye, suggesting that necrosis is a primary
mechanism of cell death in DPCs treated with these ingredients.
• Compared to CATC and pea extract, calendula extract, MI, and red clover extract detected a
modest increase in necrosis. Furthermore, apoptosis and necrosis were undetectable in DPCs
treated with CAPB, MCI, and polysorbate 60. Given the acute cytotoxicity observed with these
ingredients, additional tests may be necessary to determine the mode of cytotoxicity.
The MTS assay measures cellular metabolic activity as an indicator of cell viability. However, MTS
assessment of cell viability does not directly correlate with cell death; nor does it indicate whether cells
are undergoing apoptosis or necrosis. For example, cell growth arrest (e.g., quiescence, senescence)
can also result in an overall reduction of cell viability. Therefore, annexin V binding was measured as
an indicator of apoptosis, and the binding of DNA dye to dead cells’ DNA was measured as a necrosis
indicator.
Apoptosis was detected in DPCs treated with guar, lavender oil, or rosemary extract, while calendula
extract, CATC, and pea extract primarily induced necrosis.
Concentration-dependent increases in necrosis were detectable in DPCs treated with red clover
extract. However, >2-fold increases were detectable only at the highest concentration tested (10%),
and necrosis signals remained relatively constant, irrespective of the treatment durations. Given that
red clover-induced cytotoxicity in DPCs was apparent when cells were treated >72h at concentrations
>8%, further optimization of the assay condition may be needed.
108
FDA-CU Final
Despite the acute cytotoxicity observed with CAPB, MCI, MI, and polysorbate 60 within 24h, apoptosis
and necrosis were undetectable even as early as 15h.
In addition to apoptosis and necrosis, there exist many forms of cell death modalities, including
autophagy, anoikis, necroptosis, and ferroptosis, to mention a few [77]. While the type, intensity, and
duration of stimuli determine the mode of cell death, a drug at the same concentration can induce
multiple modes of cell death at the same time in different subpopulations of cells. Apoptosis and
necrosis can also occur independently, sequentially, and simultaneously [76]. Therefore, additional
studies that measure ATP and LDH levels, caspase activities, and/or other methods, such as flow
cytometry analysis, are required to discern the mechanisms of cell death induced by these ingredients.
Solubility
For ingredients with limited solubility, it was not feasible to test higher concentrations. For example,
guar, caused the solution to gel at concentrations higher than 0.1%. Coconut oil, olus oil, pequi oil, and
polysorbate 60 needed to be heated to 40°C. Limited solubility may cause ingredients to precipitate or
aggregate, leading to an underestimation of their activity.
Apoptosis detection
While the annexin V binding to PS is widely accepted as an indicator of apoptosis, several drawbacks
must be considered when interpreting data. PS externalization has been detected under
patho/physiological conditions unrelated to apoptosis (e.g., platelet activation), and cell swelling can
also occur in response to established apoptotic stimuli [78]. In addition, PS externalization is also
known to occur during limited windows of time, which can vary depending on the cell type,
concentration, and experimental conditions. Therefore, multiplexing using additional markers of
apoptosis, such as caspase activation, or a molecular imaging probe capable of detecting apoptosis
during a wider window of time can help confirm the occurrence of apoptosis.
109
FDA-CU Final
as these models lack the complex tissue organization and close circuitry linkage of one cell type with
the other. Therefore, while in vitro assessments inform further investigations into the properties of an
ingredient, the current study does not provide sufficient information to imply an in vitro–in vivo
correlation.
Future in vivo long-term treatment studies to assess for alopecia should include a sufficient number of
animals to statistically power the study. In addition, transcriptomic and cytokine profiling of DevaCurl-
and WEN-treated skin may help identify molecular signatures and inflammatory responses that
contribute to aberrant hair cycling associated with these products.
7 References
1. Hughes, E.C.T., A., Telogen Effluvium. StatPearls, 2018.
2. Alessandrini, A., et al., Common causes of hair loss - clinical manifestations, trichoscopy and
therapy. J Eur Acad Dermatol Venereol, 2021. 35(3): p. 629-640.
3. Asghar, F., et al., Telogen Effluvium: A Review of the Literature. Cureus, 2020. 12(5): p. e8320.
4. Zirwas, M.J., Contact Dermatitis to Cosmetics. Clin Rev Allergy Immunol, 2019. 56(1): p. 119-128.
5. Lazzarini, R., et al., Allergic contact dermatitis by shampoo components: a descriptive analysis of
20 cases. An Bras Dermatol, 2020. 95(5): p. 658-660.
6. Jacob, S.E. and S. Amini, Cocamidopropyl betaine. Dermatitis, 2008. 19(3): p. 157-60.
7. Presley, C.L., et al., The History of Surfactants and Review of Their Allergic and Irritant Properties.
Dermatitis, 2021. 32(5): p. 289-297.
8. Zirwas, M. and J. Moennich, Shampoos. Dermatitis, 2009. 20(2): p. 106-10.
9. Schneider, M.R., R. Schmidt-Ullrich, and R. Paus, The Hair Follicle as a Dynamic Miniorgan.
Current Biology, 2009. 19(3): p. R132-R142.
10. Orăsan, M.C., Andrei Evaluation of Animal Models Suitable for Hair Research and Regeneration,
in Experimental Animal Models of Human Diseases, B. Ibeh, Editor. 2017, IntechOpen. p. 235-255.
11. Nakamura, M., et al., Mutant laboratory mice with abnormalities in hair follicle morphogenesis,
cycling, and/or structure: An update. Journal of Dermatological Science, 2013. 69(1): p. 6-29.
12. Oh, J.W., et al., A Guide to Studying Human Hair Follicle Cycling In Vivo. Journal of Investigative
Dermatology, 2016. 136(1): p. 34-44.
13. Porter, R.M., Mouse models for human hair loss disorders. Journal of anatomy, 2003. 202(1): p.
125-131.
14. Castro, A.R., C. Portinha, and E. Logarinho, The Emergent Power of Human Cellular vs Mouse
Models in Translational Hair Research. Stem Cells Translational Medicine, 2022. 11(10): p. 1021-
1028.
15. Meda Sandra, O. and C. Andrei, Evaluation of Animal Models Suitable for Hair Research and
Regeneration, in Experimental Animal Models of Human Diseases, B. Ibeh, Editor. 2017,
IntechOpen: Rijeka. p. Ch. 12.
16. Hendrix, S., et al., A Guide to Assessing Damage Response Pathways of the Hair Follicle: Lessons
From Cyclophosphamide-Induced Alopecia in Mice. Journal of Investigative Dermatology, 2005.
125(1): p. 42-51.
17. Sundberg, J.P., E.M.J. Peters, and R. Paus, Analysis of Hair Follicles in Mutant Laboratory Mice.
Journal of Investigative Dermatology Symposium Proceedings, 2005. 10(3): p. 264-270.
18. Orasan, M.S., et al., Hair loss and regeneration performed on animal models. Clujul medical
(1957), 2016. 89(3): p. 327-334.
19. Müller-Röver, S., et al., A Comprehensive Guide for the Accurate Classification of Murine Hair
Follicles in Distinct Hair Cycle Stages. Journal of Investigative Dermatology, 2001. 117(1): p. 3-15.
20. Botchkarev, V.A. and A.A. Sharov, Modeling Chemotherapy-Induced Hair Loss: From Experimental
Propositions toward Clinical Reality. Journal of Investigative Dermatology, 2016. 136(3): p. 557-
559.
110
FDA-CU Final
21. Yoon, J.-S., et al., Development of a Model for Chemotherapy-Induced Alopecia: Profiling of
Histological Changes in Human Hair Follicles after Chemotherapy. Journal of Investigative
Dermatology, 2016. 136(3): p. 584-592.
22. Chen, S.-S., et al., Preventive effects of cedrol against alopecia in cyclophosphamide-treated mice.
Environmental Toxicology and Pharmacology, 2016. 46: p. 270-276.
23. Messenger, A.G.R.J., Minoxidil: mechanisms of action on hair growth. British journal of
dermatology (1951), 2004. 150(2): p. 186-194.
24. Arck, P.C., et al., Topical minoxidil counteracts stress-induced hair growth inhibition in mice.
Experimental Dermatology, 2003. 12(5): p. 580-590.
25. Chen, C.-H., et al., Simultaneous effects of tocopheryl polyethylene glycol succinate (TPGS) on
local hair growth promotion and systemic absorption of topically applied minoxidil in a mouse
model. International Journal of Pharmaceutics, 2005. 306(1): p. 91-98.
26. Jing, J., et al., Expression of decorin throughout the murine hair follicle cycle: hair cycle
dependence and anagen phase prolongation. Experimental Dermatology, 2014. 23(7): p. 486-491.
27. Jindo, T., et al., Local Injection of Hepatocyte Growth Factor/Scatter Factor (HGF/SF) Alters Cyclic
Growth of Murine Hair Follicles. Journal of Investigative Dermatology, 1998. 110(4): p. 338-342.
28. Ito, N., et al., Corticotropin-Releasing Hormone Stimulates the In Situ Generation of Mast Cells
from Precursors in the Human Hair Follicle Mesenchyme. Journal of Investigative Dermatology,
2010. 130(4): p. 995-1004.
29. Bertolini, M., et al., Abnormal interactions between perifollicular mast cells and CD8+ T-cells may
contribute to the pathogenesis of alopecia areata. PLoS One, 2014. 9(5): p. e94260.
30. Sundberg, J.P., et al., Skin Diseases in Laboratory Mice: Approaches to Drug Target Identification
and Efficacy Screening. Methods in molecular biology (Clifton, N.J.), 2016. 1438: p. 199-224.
31. Tobin, D.J., et al., The fate of hair follicle melanocytes during the hair growth cycle. J Investig
Dermatol Symp Proc, 1999. 4(3): p. 323-32.
32. Slominski, A., et al., Hair follicle pigmentation. J Invest Dermatol, 2005. 124(1): p. 13-21.
33. Geyfman, M., et al., Resting no more: re-defining telogen, the maintenance stage of the hair
growth cycle. Biological reviews of the Cambridge Philosophical Society, 2015. 90(4): p. 1179-
1196.
34. Zhao, J., et al., Suppression of FGF5 and FGF18 Expression by Cholesterol-Modified siRNAs
Promotes Hair Growth in Mice. Frontiers in Pharmacology, 2021. 12.
35. Botchkarev, V.A., Molecular Mechanisms of Chemotherapy-Induced Hair Loss. Journal of
Investigative Dermatology Symposium Proceedings, 2003. 8(1): p. 72-75.
36. Higgins, C.A., G.E. Westgate, and C.A. Jahoda, From telogen to exogen: mechanisms underlying
formation and subsequent loss of the hair club fiber. J Invest Dermatol, 2009. 129(9): p. 2100-8.
37. Arck, P.C., et al., Stress inhibits hair growth in mice by induction of premature catagen
development and deleterious perifollicular inflammatory events via neuropeptide substance P-
dependent pathways. The American journal of pathology, 2003. 162(3): p. 803-814.
38. Meyer, L., J. Caston, and A.G. Mensah-Nyagan, Seasonal variation of the impact of a stressful
procedure on open field behaviour and blood corticosterone in laboratory mice. Behavioural Brain
Research, 2006. 167(2): p. 342-348.
39. Bind, R.H., et al., The role of pheromonal responses in rodent behavior: future directions for the
development of laboratory protocols. Journal of the American Association for Laboratory Animal
Science : JAALAS, 2013. 52(2): p. 124-129.
40. Eichmüller, S., et al., Clusters of Perifollicular Macrophages in Normal Murine Skin: Physiological
Degeneration of Selected Hair Follicles by Programmed Organ Deletion. Journal of Histochemistry
& Cytochemistry, 1998. 46(3): p. 361-370.
41. Grace, S.A., et al., Presence of Mast Cells and Mast Cell Degranulation in Scalp Biopsies of
Telogen Effluvium. Int J Trichology, 2017. 9(1): p. 25-29.
42. Ribatti, D., The Staining of Mast Cells: A Historical Overview. Int Arch Allergy Immunol, 2018.
176(1): p. 55-60.
111
FDA-CU Final
43. Randolph, M. and A. Tosti, Oral minoxidil treatment for hair loss: A review of efficacy and safety.
Journal of the American Academy of Dermatology, 2021. 84(3): p. 737-746.
44. Gupta, K. and I.T. Harvima, Mast cell-neural interactions contribute to pain and itch. Immunol Rev,
2018. 282(1): p. 168-187.
45. Leon, A., et al., Itching for an answer: A review of potential mechanisms of scalp itch in psoriasis.
Exp Dermatol, 2019. 28(12): p. 1397-1404.
46. Castellana, D., R. Paus, and M. Perez-Moreno, Macrophages contribute to the cyclic activation of
adult hair follicle stem cells. PLoS Biol, 2014. 12(12): p. e1002002.
47. Wang, E.C.E., et al., A Subset of TREM2(+) Dermal Macrophages Secretes Oncostatin M to
Maintain Hair Follicle Stem Cell Quiescence and Inhibit Hair Growth. Cell Stem Cell, 2019. 24(4):
p. 654-669.e6.
48. Chen, J., et al., Mice expressing a mutant Krt75 (K6hf) allele develop hair and nail defects
resembling pachyonychia congenita. J Invest Dermatol, 2008. 128(2): p. 270-9.
49. Baroli, B., Penetration of nanoparticles and nanomaterials in the skin: Fiction or reality? Journal of
Pharmaceutical Sciences, 2010. 99(1): p. 21-50.
50. Liu, N., et al., Chronic Restraint Stress Inhibits Hair Growth via Substance P Mediated by Reactive
Oxygen Species in Mice. PLOS ONE, 2013. 8(4): p. e61574.
51. Bind, R.H., et al., The role of pheromonal responses in rodent behavior: future directions for the
development of laboratory protocols. J Am Assoc Lab Anim Sci, 2013. 52(2): p. 124-9.
52. Nguyen, S.T., H.T.-L. Nguyen, and K.D. Truong, Comparative cytotoxic effects of methanol,
ethanol and DMSO on human cancer cell lines. Biomedical Research and Therapy, 2020. 7: p.
3855-3859.
53. Bodó, E., et al., Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in
vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy. Am J
Pathol, 2007. 171(4): p. 1153-67.
54. Luanpitpong, S., et al., Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle
dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism. Apoptosis, 2011.
16(8): p. 769-82.
55. Loing, E., et al., A new strategy to modulate alopecia using a combination of two specific and
unique ingredients. J Cosmet Sci, 2013. 64(1): p. 45-58.
56. Lueangarun, S. and R. Panchaprateep, An Herbal Extract Combination (Biochanin A, Acetyl
tetrapeptide-3, and Ginseng Extracts) versus 3% Minoxidil Solution for the Treatment of
Androgenetic Alopecia: A 24-week, Prospective, Randomized, Triple-blind, Controlled Trial. J Clin
Aesthet Dermatol, 2020. 13(10): p. 32-37.
57. Dinda, M., et al., PI3K-Mediated Proliferation of Fibroblasts by Calendula officinalis Tincture:
Implication in Wound Healing. Phytotherapy Research, 2015. 29(4): p. 607-616.
58. Hormozi, M., et al., Calendula officinalis stimulate proliferation of mouse embryonic fibroblasts via
expression of growth factors TGFβ1 and bFGF. Inflamm Regen, 2019. 39: p. 7.
59. Cruceriu, D., O. Balacescu, and E. Rakosy, Calendula officinalis: Potential Roles in Cancer
Treatment and Palliative Care. Integrative cancer therapies, 2018. 17(4): p. 1068-1078.
60. Tabatabaei, M.H., et al., Cytotoxicity of the Ingredients of Commonly Used Toothpastes and
Mouthwashes on Human Gingival Fibroblasts. Frontiers in dentistry, 2019. 16(6): p. 450-457.
61. de Oliveira, M.L., et al., In vivo topical anti-inflammatory and wound healing activities of the fixed oil
of Caryocar coriaceum Wittm. seeds. J Ethnopharmacol, 2010. 129(2): p. 214-9.
62. Gao, T. and A. Bedell, Ultraviolet damage on natural gray hair and its photoprotection. J Cosmet
Sci, 2001. 52(2): p. 103-18.
63. Melin, V.E., et al., Exposure to common quaternary ammonium disinfectants decreases fertility in
mice. Reprod Toxicol, 2014. 50: p. 163-70.
64. Klimek-Szczykutowicz, M., A. Szopa, and H. Ekiert, Citrus limon (Lemon) Phenomenon-A Review
of the Chemistry, Pharmacological Properties, Applications in the Modern Pharmaceutical, Food,
112
FDA-CU Final
and Cosmetics Industries, and Biotechnological Studies. Plants (Basel, Switzerland), 2020. 9(1): p.
119.
65. Burnett, C.L., et al., Safety Assessment of Citrus-Derived Peel Oils as Used in Cosmetics. Int J
Toxicol, 2019. 38(2_suppl): p. 33s-59s.
66. Rodriguez-Homs, L.G. and A.R. Atwater, Allergens in Medical Hand Skin Cleansers. Dermatitis,
2019. 30(6).
67. Collis, R.W. and D.M. Sheinbein, Cocamidopropyl betaine is commonly found in hypoallergenic
personal care products for children. Journal of the American Academy of Dermatology, 2020.
82(5): p. 1245-1247.
68. Wallace, T.C., Health Effects of Coconut Oil-A Narrative Review of Current Evidence. J Am Coll
Nutr, 2019. 38(2): p. 97-107.
69. Rele, A.S. and R.B. Mohile, Effect of mineral oil, sunflower oil, and coconut oil on prevention of hair
damage. J Cosmet Sci, 2003. 54(2): p. 175-92.
70. Lee, B.H., J.S. Lee, and Y.C. Kim, Hair Growth-Promoting Effects of Lavender Oil in C57BL/6
Mice. Toxicological research, 2016. 32(2): p. 103-108.
71. Bilal, M. and H.M.N. Iqbal, An insight into toxicity and human-health-related adverse consequences
of cosmeceuticals — A review. Science of The Total Environment, 2019. 670: p. 555-568.
72. Herman, A., et al., Isothiazolinone derivatives and allergic contact dermatitis: a review and update.
Journal of the European Academy of Dermatology and Venereology, 2019. 33(2): p. 267-276.
73. Dhariwala, M.Y. and P. Ravikumar, An overview of herbal alternatives in androgenetic alopecia.
Journal of Cosmetic Dermatology, 2019. 18(4): p. 966-975.
74. Murata, K., et al., Promotion of Hair Growth by Rosmarinus officinalis Leaf Extract. Phytotherapy
Research, 2013. 27(2): p. 212-217.
75. Review, C.I., Safety Assessment of Rosmarinus Officinalis (Rosemary)-Derived Ingredients as
Used in Cosmetics (https://www.cir-safety.org/sites/default/files/rosmarinus_2.pdf). 2014.
76. Elmore, S., Apoptosis: a review of programmed cell death. Toxicol Pathol, 2007. 35(4): p. 495-516.
77. Yan, G., M. Elbadawi, and T. Efferth, Multiple cell death modalities and their key features (Review).
World Acad Sci J, 2020. 2(2): p. 39-48.
78. Helm, K., et al., <b><i>In Vitro</i></b> Cell Death Discrimination and Screening Method by Simple
and Cost-Effective Viability Analysis. Cellular Physiology and Biochemistry, 2017. 41(3): p. 1011-
1019.
113