Available online 21 March 2022
0887-2333/© 2022 Elsevier Ltd. All rights reserved.
Strategies for the evaluation of the eye irritation potential of different types of surfactants and silicones used in cosmetic products
Larissa de Lima S ´ a
a, Rodrigo Vieira Rodrigues
b, Vani M. Alves
c, Lorena Rigo Gaspar
a,*aSchool of Pharmaceutical Sciences of Ribeir˜ao Preto, University of S˜ao Paulo, Ribeir˜ao Preto, SP, Brazil
bInvitrocell, Investiga Research Institute, Paulínia, SP, Brazil
cFaculty of Medicine of Ribeir˜ao Preto, University of S˜ao Paulo, Brazil
A R T I C L E I N F O Editor: P Jennings Keywords:
Eye irritation HET-CAM BCOP Histopathology Surfactants Alternative method
A B S T R A C T
The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as “severe irritants”, it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as “no prediction can be made”. However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.
1. Introduction
Cosmetic products are freely available to the consumer and should be safe for human health when applied under normal or reasonable con- ditions of use (SCCS, 2016). For many years, skin and eye irritation potential was determined in experimental animals using the Draize test (Draize et al., 1944). However, due to ethical issues involving the use of experimental animals and the lack of reproducibility, over the last de- cades there have been major investments in the development and vali- dation of alternative in vitro methods, which aim to predict the responses to ocular irritation observed in the Draize test (ICCVAM, 2006a, 2006b; OECD, 2017a; Lotz et al., 2016).
To ensure consumer safety, all chemicals must be labeled with their hazard classes and categories as well as other relevant information on a material safety data sheet (MSDS) based on the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) developed by the United Nations (UN). In the GHS system, chemicals are classified according to the Draize eye test as follows (UN, 2015): Category 1 for corrosive or severe irritation to eyes (irreversible eye effects), Category 2/2A for irritation to eyes (reversible eye effects within 21 days), Category 2B for mildly irritation to eyes (reversible eye effects within 7 days) and No category (non-irritant).
Eye irritation usually involves more than one mechanism of toxicity, and an in vitro test that studies only one or two endpoints is not
Abbreviations: GHS, Globally Harmonized System of Classification and Labeling of Chemicals; OECD, Organisation for Economic Co-operation and Development;
IATA, Integrated Approach on Testing and Assessment; MSDS, Material Safety Data Sheet; HET-CAM, Hen’s Egg Test – Chorioallantoic Membrane; CAM, Chorio Allantoic Membrane; BCOP, Bovine Corneal Opacity and Permeability; SDS, Sodium Dodecyl Sulfate; SLES, Sodium Laureth Sulfate; SCG, Sodium Cocoyl Glutamate;
CCMG, Capryloyl/Caproyl Methyl Glucamide; CAB, Cocoamidopropyl Betaine; NaCl, Sodium Chloride; MSc, Mean Score; HBSS, Hanks’ Balanced Salt Solution;
EMEM, Eagle’s Minimum Essential Medium; IVIS, In vitro Irritancy Score; HE, Hematoxylin and Eosin; SCCS, Scientific Committee on Consumer Safety.
* Corresponding author at: School of Pharmaceutical Sciences of Ribeir˜ao Preto, University of S˜ao Paulo, FCFRP-USP, Av do Caf´e s/n., Bairro Monte Alegre, Ribeir˜ao Preto, SP, Brazil.
E-mail address: [email protected] (L.R. Gaspar).
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
https://doi.org/10.1016/j.tiv.2022.105351
Received 16 April 2021; Received in revised form 28 February 2022; Accepted 17 March 2022
observing the irritant effects on it immediately after the application of the pure or diluted chemical (Luepke, 1985; Luepke and Kemper, 1986).
HET-CAM is recommended by some regulatory agencies for the identi- fication of serious eye damage although it is still not recognized by the OECD as an alternative to animal experimentation due to the lack of data in the mild/moderate irritancy range (IATA 263) (OECD, 2017a; ECHA, 2015). Therefore, this assay is also being suggested in the bottom-up OECD strategy for the evaluation of ocular toxicity potential in order to increase the prediction specificity and sensitivity (IATA 263) (OECD, 2017a). In fact, it is the only in vitro alternative method that addresses vascular events. It provides hyperemia, hemorrhage and coagulation data that are similar to those of animal conjunctiva (ICCVAM, 2006b) and has some advantages such as being simple, fast, easy to handle and relatively inexpensive.
The Bovine Corneal Opacity and Permeability (BCOP) assay is an organotypic model (Chamberlain et al., 1997) that uses isolated corneas from bovines. The evaluated substances are applied to the surface of the epithelium and the damages caused by them are evaluated by changes in corneal opacity and permeability (OECD TG 437) (OECD, 2020). Both measurements are used to calculate the irritation score, which is used to classify the degree of in vitro ocular irritation of the chemical tested in order to predict its potential for eye irritation in vivo (OECD, 2020). In addition, histopathological evaluation of the corneas may be a useful tool by identifying corneal layer damage that does not show significant values of opacity and/or permeability. It allows the assessment of the degree and depth of damage (OECD, 2017b), as well as the discrimi- nation between mild and moderate irritants (Cater and Harbell, 2006).
Surfactants are important components in the formulation of cosmetic and hygiene products such as shampoos, soaps, and lotions. Despite being classified as irritants (categories 1 and 2), they are used in rinse-off products as diluted solutions (from 10 to 50%), which are considered safe for consumers, and an assay for predicting the different irritation scores provoked by these different concentrations is also very important prior to clinical assays. Thus, three anionic surfactants, two non-ionic surfactants and one amphoteric surfactant were evaluated here at different dilutions. Moreover, silicones widely used in the manufacture of hair, face, and body products, mainly for imparting dry touch, soft- ness and a pleasant sensorial feel after the application of the product (Rieger, 2000) were also evaluated.
Thus, the present study proposed to apply two strategies for the prediction of the eye irritation potential of non-irritants by using BCOP, followed by histopathological evaluation of corneas exposed to the test substances and the other by using HET-CAM for the evaluation of different concentrations of surfactants and silicones. The present study also shows the importance of HET-CAM events and histopathological analysis as an in vitro strategy to be used to differentiate and select the safer concentration of surfactants to be applied in cosmetics. These test strategies could precede clinical assays and consequently reduce
exposure of humans to high irritant formulations.
2. Material and methods 2.1. Chemicals
Anionic surfactants: sodium dodecyl sulfate (SDS, CAS 151–21-3, Synth, Brazil; 90% purity), sodium laureth sulfate (SLES, CAS 1335-72- 4, Synth, Brazil; 27% purity), sodium cocoyl glutamate (SCG, CAS 68187–32-6, Hostapon CCG®, Clariant, Brazil; 33% purity). Non-ionic surfactants: polysorbate 20 (CAS 9005-64-5, Tween®20, USB Corpora- tion, USA), capryloyl/caproyl methyl glucamide (CCMG, CAS 1591782–62-5, GlucoTain®Clear, Clariant, Germany; 50% purity).
Amphoteric surfactant: cocamidopropyl betaine (CAB, CAS 61789–40-0, Genagen®CAB-CM, Clariant, Brazil; 30% purity). Silicones: dimethicone (CAS 63148–62-9, Xiameter®PMX-200 Silicone Fluid, 50 CS, Dow Corning, USA), cyclopentasiloxane (CAS 541–02-6, Xiameter®PMX- 245, Dow Corning, UK).
2.2. Approaches for assessing eye irritation
All surfactants and silicones were evaluated by both strategies: HET- CAM assay (Luepke and Kemper, 1986) and also by BCOP test (OECD 437, 2020) followed by histopathological analysis by OECD GD 160 (OECD, 2017b) in order to evaluate the range of UN GHS eye irritation classifications, The test substances are presented in Table 1 and the methods are described in items 2.3 to 2.5.
2.3. HET-CAM
The HET-CAM assay was performed according to Luepke and Kemper (1986). Fertilized chicken eggs from White Leghorn species were incu- bated for 10 days at 37 ◦C under 50–60% relative humidity in an incubator. On the 10th day of incubation, the piece of eggshell covering the air space part was removed and the eggshell membrane was hy- drated with 300 μL 0.9% NaCl and removed carefully and the chorio- allantoic membrane (CAM) was exposed. Three hundred μL of each negative (0.9% NaCl) and positive (1% SDS) control and test substances were applied to the CAM as shown in Table 1.
After 20 s, the CAM was rinsed off with 5 mL 0.9% NaCl and vascular events such as hyperemia (Hyp), hemorrhage (Hem) and coagulation (Coa) were recorded for 5 min using an USB microscope (DigiMicro2.0 Scale, Germany). The assay was carried out on four eggs in two inde- pendent assays. The irritancy score or mean score (MSc) was calculated according to Eq. (1).
where Hyp, Hem and Coa are the times in seconds corresponding to the onset of hyperemia, hemorrhage and coagulation, respectively. The irritation categories were considered according to Luepke and Kemper (1986): non-irritant (MSc <1), mild irritant (1 ≤MSc <5), moderate irritant (5 ≤MSc <9), and severe irritant (MSc ≥9).
2.4. BCOP
The BCOP assay was performed according to OECD TG 437 (OECD, 2020). Briefly, eyes were collected at a slaughterhouse (Cow Pig, Boi- tuva – SP, Brazil) and immersed completely in cooled Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and strepto- mycin at 100 μg/mL. Isolated corneas were mounted in holders and filled with Eagle’s Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ±1 ◦C for 1 h and the baseline opacity of each cornea was read on an opacitometer (OP-KIT, Electro Design, France).
The corneas were exposed to negative and positive controls (0.9% NaCl and 100% ethanol, respectively) and to test substances prepared in sa- line solution (0.9% NaCl) or undiluted, as shown in Table 1.
After 10 min of exposure, test substances and controls were removed from the anterior chamber and the epithelium was washed. After 2 h of incubation of the holders with EMEM, corneal effects were measured by decreased light transmission (corneal opacity) using the opacitometer.
Then, 1 mL of sodium fluorescein solution (4 mg/mL) was added to the anterior chamber, which interfaces with the epithelial side of the cornea, while the posterior chamber, which interfaces with the endothelial side of the cornea, was filled with fresh EMEM. The holder was incubated for 90 min at 32 ±1 ◦C. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with a spectropho- tometer UV/VIS (SpectraMax M5, Molecular Devices, USA) at 490 nm (OD490). The mean opacity and permeability (OD490) values for each treatment group were combined to obtain an In vitro Irritancy Score (IVIS) as follows Eq. (2) (OECD, 2020):
IVIS=mean opacity value+ (15×mean permeability OD490value) (2) Each treatment group IVIS was analyzed and classified according to the ocular irritation potential described in OECD TG 437: no category (IVIS ≤3), no prediction can be made (IVIS >3; ≤55), category 1 (IVIS
>55).
2.5. Histopathological analysis of BCOP corneas
Histopathological analysis was performed as suggested by OECD GD
160 (OECD, 2017b). After the BCOP assay, corneas were fixed in 10%
phosphate buffered formalin solution at room temperature. The corneas were bisected, dehydrated in graded ethanol (70–100%), cleared in xylene, embedded in paraffin, sectioned at 5 μm using a microtome (Leica RM 2065, Germany) and stained with hematoxylin and eosin (HE). Histological slices were analyzed using a microscope (Leica DM 5000 B, Germany) at 20 magnification and the photographic represen- tations (Leica DFC 295 camera, Germany) of the corneas for each exposed test substance and control group were prepared and analyzed with LAS 4.0 software. Histopathological changes of the corneal epithelium, stroma, and endothelium were verified, and the irritation potential of each test substance was classified according to the location of lesions provoked in the epithelium, as well as lesions caused in the stroma based on the extension of loose collagen bundles and swelling of the keratocytes (Table 2) (Furukawa et al., 2015).
2.6. Statistical analysis
Score values of opacity and permeability from BCOP, and hyperemia, hemorrhage, coagulation, and mean scores from the HET-CAM assay showed normal distribution in the Anderson-Darling Normality Test.
Therefore, they were submitted to statistical analysis by Analysis of Variance (ANOVA) followed by post-hoc Tukey analysis. Values of p <
0.05 were considered significant. All calculations were performed using Minitab 17.
3. Results 3.1. HET-CAM
Initially, the events induced by the negative (0.9% NaCl) and posi- tive (1% SDS) controls on the CAM were evaluated (Fig. 1) and the MSc were also calculated. As expected, 0.9% NaCl did not cause any irritation event; 1% SDS caused mainly hemorrhage on the CAM, which was characterized by small bleeding (Fig. 1) with an MSc of 12.00 and was classified as a severe irritant (Table 3). These data were similar to rec- ommended values (ICCVAM, 2006b).
Regarding the anionic surfactant SLES, all studied SLES concentra- tions (27%, 10%, 7% and 1%) were considered to be severe irritants (MSc ≥9) (Fig. 2). 7% SLES showed intermediate MSc (15.50) and was statistically equivalent to 27% (17.75) and 10% (15.25) SLES but 27%
SLES was statistically different from 10% SLES (p <0.001). All these results were considered statistically higher than 1% SLES (MSc: 9.00) (p
<0.001). The difference observed among these MSc values was mainly due to coagulation scores, since 1% SLES did not provoke any coagu- lation on CAM (mean coagulation score: 0), a value that was statistically lower than that of 27%, 10% and 7% SLES, which provoked coagulation on CAM (p <0.001) (mean coagulation scores: 7.00, 5.25 and 5.00, respectively). The hemorrhage (27%: 6.50, 10%: 5.50, 7%: 6.25 and 1%:
5.50) and hyperemia (27%: 4.25, 10%: 4.50, 7%: 4.25 and 1%: 3.50) scores of the four concentrations analyzed were considered statistically equivalent (p > 0.05) (Table 3). The difference in the intensity of vascular reactions was also observed qualitatively (Fig. 2). The anionic surfactant SDS 10% was also considered to be severe irritant showing the highest MSc (21.00) and coagulation scores (9.00), which were statistically higher than all analyzed substances.
The other analyzed anionic surfactant sodium cocoyl glutamate (SCG) showed an MSc of 12.00 at both studied concentrations and was classified as a severe irritant (Table 3). The Cosmetic Ingredient Review Table 2
Classification of the irritation potential of a chemical according to the depth of lesions detected in bovine corneal epithelium and stroma.
Irritation
categories Location of lesions on
the corneal epithelium Location of lesions on the corneal stroma
Non-irritant Absent Absent
Slight irritant Squamous cells Absent Mild irritant Between the squamous
and upper wing cells Matrix damage extending no further than the upper third
Moderate
irritant From squamous to
under wing cells Matrix damage extending no further than two-thirds
Severe
irritant Basal cells Matrix damage extending into the lower third of the corneal stroma and/
or causing damage to the endothelial cells
MSc=(Hyp1±Hem1±Coa1) ± (Hyp2±Hem2±Coa2) ± (Hyp3±Hem3±Coa3) ± (Hyp4±Hem4±Coa4)
4 (1)
(Burnett et al., 2017) also obtained the same classification for SCG in the HET-CAM test. The two studied concentrations did not provoke any coagulation on CAM (mean coagulation score: 0.00) as observed for 1%
SLES; however, their MSc was considered statistically higher than that of 1% SLES (Table 3, Fig. 3) due to higher hyperemia and hemorrhage scores. The same values were obtained for the non-ionic surfactant capryloyl/caproyl methyl glucamide (CCMG) at 3% and 1.5% (Table 3).
The difference between the two surfactant concentrations studied could be observed only qualitatively in Fig. 4, where different intensities of hemorrhage can be seen.
When the non-ionic surfactant polysorbate 20 (at 10%) was applied to CAM, it induced mild signs of bleeding (Fig. 4) and was classified as a moderate irritant with an MSc of 5.62 (Table 3). This result (MSc) and
hyperemia scores were considered statistically lower (p <0.001) than those of the positive control (1% SDS) and of the other surfactants studied.
The amphoteric surfactant CAB was also considered to be a severe irritant, especially at 3% concentration, which showed one of the highest MSc values (MSc: 19.00) and was statistically higher than the value of 1.5% CAB (MSc: 16.25) (Table 3) (p <0.001). This difference was mainly due to coagulation score since 3% CAB caused more intense and statistically significant coagulation (mean coagulation score: 7.00) than 1.5% CAB (mean coagulation score: 4.25) (p <0.001) (Table 3).
This difference in coagulation could also be observed qualitatively, since hemorrhage and coagulation were observed on CAM treated with 3%
CAB (Fig. 5), whereas 1.5% CAB caused hemorrhage and discrete Fig. 1. HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the negative (0.9% NaCl) and positive (1% SDS) controls. Arrows show hemorrhage (↑).
Table 3
HET-CAM assay: mean score of hyperemia, hemorrhage, coagulation, and mean score (MSc), standard deviation (SD) and irritation category of the test substances.
Quadruplicates from two independent experiments resulting in n =8 samples per group.
Test substances Mean hyperemia score ±DP Mean hemorrhage score ±DP Mean coagulation score ±DP MSc ±SD Irritation category
0.9% NaCl 0.00 ±0,00 ■ 0.00 ±0.00 ♥ 0.00 ±0.00 α 0.00 ±0.00H Non-irritant*
10% SDS 5.00 ±0.00 ● 7.00 ±0.00 ◆ 9.00 ±0.00 μ 21.00 ±0.00 A Severe irritant***
1% SDS 5.00 ±0.00 ● 7.00 ±0.00 ◆ 0.00 ±0.00 α 12.00 ±0.00 E Severe irritant***
27% SLES 4.25 ±1.03 ● ▴ 6.50 ±0.93 ♣◆ 7.00 ±0.00 β 17.75 ±1.83 BC Severe irritant***
10% SLES 4.50 ±0.93 ● ▴ 5.5 ±0.93 ♣♠ 5.25 ±0.71 δ 15.25 ±1.28 D Severe irritant***
7% SLES 4.25 ±1.03 ● ▴ 6.25 ±1.03 ♣◆ 5.00 ±0.00 δ 15.50 ±2.07 CD Severe irritant***
1% SLES 3.50 ±0.93 ▴ 5.50 ±0.93 ♣♠ 0.00 ±0.00 α 9.00 ±1.85 F Severe irritant***
3% SCG 5.00 ±0.00 ● 7.00 ±0.00 ◆ 0.00 ±0.00 α 12.00 ±0.00 E Severe irritant***
1.5% SCG 5.00 ±0.00 ● 7.00 ±0.00 ◆ 0.00 ±0.00 α 12.00 ±0.00 E Severe irritant***
10% Polysorbate 20 1.25 ±1.49 □ 4.75 ±1.28 ♠ 0.00 ±0.00 α 5.62 ±2.26 G Moderate irritant**
3% CCMG 5.00 ±0.00 ● 7.00 ±0.00 ◆ 0.00 ±0.00 α 12.00 ±0.00 E Severe irritant***
1.5% CCMG 5.00 ±0.00 ● 7.00 ±0.00 ◆ 0.00 ±0.00 α 12.00 ±0.00 E Severe irritant***
3% CAB 5.00 ±0.00 ● 7.00 ±0.00 ◆ 7.00 ±0.00 β 19.00 ±0.00 AB Severe irritant***
1.5% CAB 5.00 ±0.00 ● 7.00 ±0.00 ◆ 4.25 ±2.76 δ 16.25 ±2.76 CD Severe irritant***
Dimethicone 0.00 ±0.00 ■ 0.00 ±0.00 ♥ 0.00 ±0.00 α 0.00 ±0.00H Non-irritant*
Cyclopentasiloxane 0.00 ±0.00 ■ 0.00 ±0.00 ♥ 0.00 ±0.00 α 0.00 ±0.00H Non-irritant*
Different symbols indicate statistically different hyperemia scores (■ ∕=● ∕=▴ ∕=□) (p <0.001). All studied SLES concentrations were considered statistically equivalent.
Different symbols indicate statistically different hemorrhage scores (♥ ∕=◆ ∕=♣ ∕=♠) (p <0.001). All studied SLES concentrations were considered statistically equivalent.
Different symbols indicate statistically different coagulation scores (α ∕=β ∕=δ ∕=μ) (p <0.001). 10% and 7% SLES showed intermediate values and were statistically equivalent to 1.5% CAB; however, 3% CAB was statistically different from 1.5% CAB and statistically equivalent to 27% SLES. 10% SDS was statistically different from other substances.
Different symbols indicate statistically different MSc values (A ∕=B ∕=C ∕=D ∕=E ∕=F ∕=G ∕=H) (p <0.001). 7% SLES showed intermediate values and was statistically equivalent to 27 and 10% SLES but 27% SLES was statistically different from 10% SLES. 10% SDS was statistically different from other substances.
*MSc ≤1, according to Luepke and Kemper (1986).
**5 ≤MSc <9, according to Luepke and Kemper (1986).
***MSc ≥9, according to Luepke and Kemper (1986).
coagulation signals (Fig. 5).
The silicones, dimethicone and cyclopentasiloxane, did not provoke any sign of irritation on CAM (Fig. 6) and therefore were considered non-irritants.
3.2. BCOP
The results for the eye irritation potential of all tested substances measured by their ability to induce opacity and increased permeability in isolated bovine corneas are shown in Table 4.
As expected, the corneas exposed to the negative control (0.9% NaCl) Fig. 2. HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the anionic surfactant Sodium Laureth Sulfate (SLES). Arrows show hemorrhage (↑) and coagulation ( ).
Fig. 3. HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the anionic surfactant sodium cocoyl glutamate (SCG). Arrows show hemorrhage (↑).
produced no changes in corneal opacity and permeability; the positive control (100% ethanol) altered both endpoints when compared to the negative control (p < 0.001). These data were similar to the values recommended by OECD TG 437 (OECD, 2020).
Only 10% SDS, 7% SLES and 3% CCMG were capable of inducing opacity in corneas (p <0.001) when compared to the negative control (0.9% NaCl). The different SLES concentrations studied showed statis- tically equivalent values of opacity in corneas, as also observed for different concentrations of CAB and SCG. Among the studied surfac- tants, only 3% CCMG showed statistically higher opacity values when compared to 1.5% CCMG and to the other surfactants studied (SLES, CAB and SCG (p <0.001). 10% SDS induced statistically higher opacity values when compared to 1% (Table 4).
Regarding permeability, besides 10% SDS, 3% CCMG and 7% SLES that also induced cornea opacity, another surfactant, 3% and 1.5% CAB, also enhanced cornea permeability to fluorescein (p <0.001) when compared to the negative control (0.9% NaCl). The different SLES concentrations studied showed statistically equivalent permeability values in the corneas, as observed for the other cornea endpoint (Table 4). The only difference considered to be statistically significant in the SLES groups was observed between the 7% and 1% SLES, which showed the highest and lowest values, respectively. Both CAB concen- trations studied (3% and 1.5%) presented statistically equivalent cornea permeability values, and the same equivalence was found for the SCG concentrations studied (3% and 1.5%). However, 3% CCMG showed statistically higher permeability values when compared to 1.5% CCMG Fig. 4.HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the non-ionic surfactants capryloyl/caproyl methyl glucamide (CCMG) and Polysorbate 20. Arrows show hemorrhage (↑).
Fig. 5.HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the amphoteric surfactant CAB. Arrows show hemorrhage (↑) and coagulation ( ).
and the same behavior was observed when 10% and 1% SDS were compared (p <0.001) (Table 4).
Regarding the final BCOP score (IVIS), most of the test substances were categorized as “no prediction can be made”, except for the positive and negative controls (0.9% NaCl and 100% ethanol), correctly classi- fied as no category and category 1, respectively; and for polysorbate 20 (10%), 1.5% CCMG and the silicones, which were classified as “no category”. 10% SDS, 10% and 7% SLES, 3% CCMG and 3% and 1.5%
CAB showed statistically higher IVIS (p <0.001) when compared to the negative control (0.9% NaCl) (Table 4). The different SLES concentra- tions studied showed statistically equivalent IVIS, as also observed for permeability and opacity. The only difference considered statistically significant in the SLES groups was observed between 7% and 1% SLES which showed the highest and lowest IVIS values, respectively, in agreement with permeability values. IVIS was statistically equivalent when 3% and 1.5% SCG were compared to each other. However, 3%
CCMG showed statistically higher IVIS values when compared to 1.5%
CCMG and the same behavior was observed when 10% and 1% SDS were compared (p <0.001) as well as when 3% and 1.5% CAB were compared to each other (Table 4).
3.3. Histopathological analysis of corneas submitted to the BCOP assay Corneas exposed to the negative control (0.9% NaCl) did not suffer any change in their epithelial cell layers or in the stromal collagen matrix (Fig. 7a), unlike the corneas exposed to the positive control (100% ethanol) which showed loss of superficial epithelial cells, squa- mous cell coagulation, nuclear and cytoplasmic vacuolization in the wing and basal cells, besides morphological changes of keratocytes of the upper first third of the stroma (Fig. 7b). Thus, the positive control was classified as a severe irritant.
Corneas in contact with 1% SDS only showed loss of epithelial cells from the squamous layer to part of the upper wing cell layer (Fig. 7c).
Thus, according to Furukawa et al. (2015), at this concentration, SDS showed a mild irritant potential. However, 10% SDS caused loss of cells from superficial layers to the basal cell layer of the corneal epithelium, and pyknotic nuclei in the basal layer were also observed (Fig. 7d).
Consequently, 10% SDS was classified as a severe or category 1 irritant (Furukawa et al., 2015). These analyses corroborate the statistically different BCOP data of opacity and permeability obtained for different SDS concentrations.
Treatment with 1% SLES caused a slight loss of squamous cells (Fig. 7e), with this test substance being considered a slight irritant (Furukawa et al., 2015). On the other hand, 7, 10 and 27% SLES caused cellular desquamation from the squamous cell layer to the wing cell Fig. 6. HET-CAM assay: chorioallantoic membrane before (0 s) and 5 min. post application of the silicones dimethicone and cyclopentasiloxane.
Table 4
BCOP assay: opacity and permeability values with their standard deviation, in vitro irritancy score (IVIS) and irritation category of test substances. N =4.
Test substances Opacity Permeability IVIS Irritation category
0.9% NaCl 0.25 ±
0.50 ■ −0.01 ±0.01 α
−0,06 ±
0.50 I No category*
100% Ethanol 27.50 ±
4.57 ● 1.97 ±0.71 β
δ 57.10 ±
6.12 A Category 1***
10% SDS 9.50 ±
1.91 ▴□ 2.25 ±0.65 β
δ 43.24 ±
9.69 A B No prediction can be made**
1% SDS 3.25 ±
2.63 ■ ◊ 0.75 ±0.41 α
μ γ ε 14.51 ± 8.41 E F G
H
No prediction can be made**
27% SLES 3.67 ±
1.53 ■ ◊ 0.74 ±0.21α
μ γ ε 14.72 ± 4.71 D E F G
H I
No prediction can be made**
10% SLES 4.67 ±
0.58 ■ ◊ 0.83 ±0.34 α
μ γ ε 17.09 ±
5.17 D E F G No prediction can be made**
7% SLES 5.00 ±
1.73 □ ◊ 1.25 ±0.31 δ
μ γ ε 23.82 ±
4.40C D E No prediction can be made**
1% SLES 0.67 ±
1.15 ■ ◊ 0.29 ±0.15 α
ε 4.99 ±
3.13 F G H I No prediction can be made**
3% SCG 0.50 ±
1.41 ■ ◊ 0.41 ±0.12 α
γ ε 6.60 ±
2.49 F G H I No prediction can be made**
1.5% SCG 0.50 ±
0.82 ■ ◊ 0.18 ±0.09 α 3.21 ±
1.14 G H I No prediction can be made**
10% Polysorbate
20 0.08 ±
0.50 ■ 0.02 ±0.02 α 0.38 ±
0.62H I No category*
3% CCMG 9.75 ±
0.96 ▴ 1.32 ±0.54 δ
μ γ 29.02 ±
9.07 B C D No prediction can be made**
1.5% CCMG 0.25 ±
1.26 ■ 0.11 ±0.04 α 1.30 ±
0.67H I No category*
3% CAB 3.0 ±
1.41 ■ ◊ 2.33 ±0.48 β 36.57 ±
11.15 B C No prediction can be made**
1.5% CAB 2.00 ±
0.00 ■ ◊ 1.48 ±0.50 β
δ μ 19.91 ±
4.29 D E F No prediction can be made**
Dimethicone 0.42 ±
0.50 ■ ◊ 0.00 ±0.00 α 0.46 ±
0.51H I No category*
Cyclopentasiloxane 0.17 ±
0.58 ■ 0.01 ±0.02 α 0.36 ±
0.73H I No category*
Different symbols indicate statistically different opacity scores (■ ∕=● ∕=▴ ∕=□
∕
=◊) (p <0.001).
Different symbols indicate statistically different permeability scores (α ∕=β ∕=δ
∕
=μ ∕=γ ∕=ε) (p <0.001).
Different symbols indicate statistically different IVIS scores (A ∕=B ∕=C ∕=D ∕=E
∕
=F ∕=G ∕=H ∕=I) (p <0.001).
*IVIS ≤3, according to OECD TG 437.
**3 <IVIS ≤55, according to OECD TG 437.
***IVIS >55, according to OECD TG 437.
Fig. 7. Photomicrographic representations of corneal cell layers and corneas exposed to the test substances in the BCOP assay. Arrows show the vacuoles (↕), pyknotic nuclei ( ) and edema (↑). HE stain. 20×Magnification.
Fig. 7. (continued).
showed one of the highest IVIS in BCOP, with swelling of epithelial cells and destruction of the basal cell layer, besides edema in the upper half of the stroma (Fig. 7l). Thus, both CAB concentrations were considered to be severe irritants (Furukawa et al., 2015).
On the other hand, corneas exposed to 10% polysorbate 20 only showed one row of superficial squamous cell desquamation (Fig. 7m) and therefore this substance was considered to be a slight irritant.
Similarly, corneas exposed to 1.5% CCMG showed only superficial squamous cell desquamation (Fig. 7n), and thus this substance was classified as a slight irritant. On the other hand, 3% CCMG provoked swelling of epithelial cells, desquamation from the squamous cells to the wing cells in a part of the epithelium and vacuoles in the basal layer (Fig. 7o), characterizing severe damage to the corneas. These CCMG results corroborate statistically different BCOP results of opacity and permeability between the two concentrations (Table 4), i.e., 1.5% CCMG induced less protein denaturation (low opacity) and permeability and therefore less corneal damage at a lower concentration.
Finally, dimethicone and cyclopentasiloxane did not cause any change in the different corneal layers (figure not shown) and therefore were considered to be non-irritants. The BCOP and HET-CAM silicone results are in line with histopathological analysis.
almost all surfactants could not be predicted and differences in the ef- fects provoked by different concentrations could not be observed, except for CCMG. We observed that most of the surfactants did not cause any change in the opacity of the corneas, but most of them altered their permeability, resulting in the ‘no prediction can be made’ classification (Table 4). The higher permeability scores could be attributed to the destruction of the corneal epithelial layer caused by surfactants (Gau- theron et al., 1992). On the other hand, opacity changes occur when the collagen matrix undergoes some modification (FURUKAWA et al., 2015). Although most surfactants penetrated the corneal layers, it can be inferred that they did not reach the stroma or, if they reached it, they did not interact with collagen fibers. However, it was not possible to spe- cifically classify the test substances by using only the BCOP test (OECD TG 437) due to its limitations (OECD, 2020). In addition, BCOP alone does not allow to classify any substance as category 2 (mild and mod- erate irritants). Thus, we performed histopathological analysis of the corneas used for BCOP, as suggested by OECD GD 160 (OECD, 2017b) and by other researches (Cooper et al., 2001; Cazelle et al., 2014; Fur- ukawa et al., 2015, 2017; Oliveira et al., 2015) in order to complement their results.
Histopathological analysis was also considered here as a very important tool to confirm the mechanism of action of the studied com- pounds and it demonstrated that most surfactants induced loss of
Table 5
Classification of the eye irritation potential of test substances according to performed assay.
Test substances HET-CAM BCOP Histopathology Final classification of the study GHS*
10% SDS Severe irritant No prediction Severe irritant Severe irritant Category 1 a
1% SDS Severe irritant No prediction Mild irritant Mild irritant Category 1 a
27% SLES Severe irritant with coagulation No prediction Moderate irritant Moderate irritant Category 2 b
10% SLES Severe irritant with coagulation No prediction Moderate irritant Moderate irritant Category 2 b
7% SLES Severe irritant with coagulation No prediction Moderate irritant Moderate irritant Category 2 b
1% SLES Severe irritant No prediction Slight irritant Slight irritant Category 2 b
3% SCG Severe irritant No prediction Mild irritant Mild irritant Category 2 c
1.5% SCG Severe irritant No prediction Non-irritant Non-irritant Category 2 c
3% CAB Severe irritant with coagulation No prediction Severe irritant Severe irritant Category 1 d
1.5% CAB Severe irritant with coagulation No prediction Severe irritant Severe irritant Category 1 d
10% Polysorbate 20 Moderate irritant No category Slight irritant Slight irritant No Category e
3% CCMG Severe irritant No prediction Severe irritant Severe irritant Category 1 f
1.5% CCMG Severe irritant No category Slight irritant Slight irritant Category 1 f
Dimethicone (100%) Non-irritant No category Non-irritant Non-irritant No category g
Cyclopentasiloxane (100%) Non-irritant No category Non-irritant Non-irritant No category g
Samples not studied.
*Based on Material Safety Data Sheet (MSDS) according to the GHS classification: Category 1: severe irritant; Category 2: irritant; No category: non-irritant.
aAccording to raw material MSDS with 90% purity.
b According to raw material MSDS with 27% purity.
cAccording to raw material MSDS with 33% purity.
dAccording to raw material MSDS with 30% purity.
eAccording to raw material MSDS with 100% purity.
fAccording to raw material MSDS with 50% purity.
gAccording to raw material MSDS with 100% purity.
corneal epithelial layers. The test substances that just induced superfi- cial or intermediate loss of epithelial layers were considered mild (Fig. 7c and j: 1% SDS and 3% SCG, respectively) or moderate (Fig. 7f, g and h: 7% SLES, 10% SLES and 27% SLES, respectively) irritants (Table 5) because the epithelial cells are in constant renewal through cell migration from basal to squamous layers, favoring the replacement of the injured cells (Furukawa et al., 2015; Furukawa et al., 2017). On the other hand, basal cells are the only ones that undergo mitosis, migrating from the inferior layer of the epithelium to the upper layers, and then differentiating into wing and squamous cells. Consequently, if they are damaged, it is possible that the epithelium will not be repaired, characterizing an irreversible lesion provoked by category 1 or severe irritants, which could be observed in corneas exposed to 10% SDS, 3%
and 1.5% CAB, and 3% CCMG (Fig. 7d, l, k and o, respectively); thus, these substances were categorized as severe irritants (Table 5). These corneas exhibited loss of epithelial and basal cells, pyknotic nuclei representing dead cells, and cytoplasmic vacuoles. The histological analysis cited above allowed their correct classification when compared to GHS, a fact that was not possible with IVIS (opacity and permeability) BCOP classification, that is, it was possible to move from ‘no prediction can be made’ to mild or moderate or severe irritant classification.
Thus, the present study proposed different strategies whereby the reactions observed to calculate MSc (hemorrhage and coagulation) and IVIS (opacity and permeability) were also compared statistically in order to differentiate these events and to propose the main mechanism of action involved in the irritation endpoint. BCOP data were com- plemented with histopathological analysis. All the results obtained are summarized in Table 5 and the final classification of the analyzed so- lutions is presented and compared to GHS category classification.
Although classified in the same irritation category, 1% SLES was discriminated from 10% and 7% SLES due to statistically different MSc in the HET-CAM assay (9.00 ∕=15.25 and 15.50) and to the absence of coagulation in CAM, which also demonstrated increased severity of eye irritation (IATA 263) (OECD, 2017a). Corroborating these results, BCOP histopathological analysis differentiated the classification of the various concentrations as causing slight versus moderate damage to the cornea.
The Cosmetic Ingredient Review published the SLES studies, with a paper showing that ocular irritation can occur in animals and humans as a consequence of the use of cosmetic formulations containing SLES, with the severity of the irritation appearing to increase with increased SLES concentrations. However, this surfactant is considered safe for use in cosmetics (Robinson et al., 2010).
The other anionic surfactant, SCG, had a statistically lower HET-CAM MSc score than SLES solutions but the difference between 3% and 1.5%
SCG was only observed by BCOP histopathological analysis of the cornea.
The non-ionic surfactant 3% and 1.5% CCMG could be differentiated by the BCOP IVIS score (29.02 and 1.30) (Table 4) and confirmed by histopathological analysis which demonstrated greater damage caused by the higher concentration (Fig. 7o and n, respectively).
It was interesting to note that 10% Polysorbate 20 and 1.5% CCMG were classified as ‘no category’ in the BCOP assay (Table 4) because they did not change corneal opacity and permeability. However, histopath- ological analysis showed some desquamation on superficial epithelial cells, characterizing these substances as slight irritants. However, this superficial damage is expected to recover rapidly and consequently the two substances do not pose a risk to the eyes (Furukawa et al., 2017).
The same result was obtained by Cooper et al. (2001) and Furukawa et al. (2015) when they studied some shampoos and make-up removers.
Concerning the amphoteric surfactant CAB, there was no difference in classification (irritation score/category) by HET-CAM (severe irri- tant) and by BCOP (no prediction can be made) between the two con- centrations studied, although HET-CAM MSc and coagulation scores and IVIS BCOP scores were considered to differ statistically between the two concentrations studied. Histopathological analysis of the cornea also showed a difference between the studied concentrations and classified
both of them as severe irritants.
No vascular event was observed in the HET-CAM, nor damage in BCOP and histopathological analysis post-treatment with silicones, which reinforces the sensitivity of HET-CAM and BCOP to non-irritating substances. These results are in line with a European Community Sci- entific Committee on Consumer Safety (SCCS) publication that consid- ered cyclopentasiloxane (SCCS, 2015) and dimethicone (Nair and Elmore, 2003) as substances with low ocular irritation potential.
Therefore, histopathological evaluation was extremely important as a complementary and more specific tool for the classification of most of the studied surfactants, which were classified as “no prediction can be made” (IVIS>3 and ≤ 55) by BCOP. The histopathology results corroborated the results of the BCOP assay, since most of the substances did not change the opacity of the corneas, indicating that there was no damage to the collagen matrix. On the other hand, some changes (desquamation) of the cellular layers of the epithelium were observed in the histological analysis, probably related to increased corneal perme- ability to fluorescein (Andrade et al., 2019).
It should be noted that the classification obtained in this study for some substances was different from their GHS classification provided by the MSDS, since the latter concerns the concentrated substance (pure or partially diluted raw material), while our study refers to samples diluted at low concentrations, similar to those used in cosmetic products.
According to the results obtained in the present study, the following strategies were proposed for the evaluation of the ocular irritation po- tential of cosmetic ingredients which are presumed to pose no risk at the concentrations used in cosmetic products (bottom-up study) (Fig. 8):
The HET-CAM assay should be used, which evaluates the vascular events of hyperemia, hemorrhage and coagulation that may occur in CAM after exposure to the ingredient, similarly to the effects observed in the ocular conjunctiva. The second strategy, all ingredients under study should be evaluated by the BCOP assay, which determines their effects on the cornea, i.e., whether they can alter corneal opacity and perme- ability. Depending on BCOP results the ingredient is evaluated by his- topathology for the degree and depth of the lesion provoked in the bovine cornea, permitting inferences about the reversibility of the lesion. This is possible because the corneal epithelium responds quickly to the injury caused by the chemical and thus requires a constant renewal of its cells. The basal cells, the only ones that undergo mitosis, migrate from the inferior layer of the epithelium to the upper layers, differentiating into wing and squamous cells. Consequently, the epithelial surface is continuously renewed or repaired when some damage occurs (Furukawa et al., 2015; Furukawa et al., 2017). We can conclude that the chance of reversibility of the damage is minor when there is a deeper injury (GHS category 1). Therefore, the chemical studied at this stage of the evaluation may be classified as a non-irritant (GHS no category) or mild, moderate (GHS category 2) or severe irritant (GHS category 1).
These are important assays since they evaluate different mechanisms of toxicity, which, according to Adriaens et al. (2014), are the two most important endpoints driving Category 2 classification, i.e., conjunctival redness (75–81%) and corneal opacity (54–75%).
HET-CAM is a cheap and simple method that involves conjunctival redness, which is an important endpoint driving Draize test Category 2 classification. Regarding HET-CAM assay (Fig. 8a).
• If the ingredient is classified as non-irritant in HET-CAM (MSc ≤0.9), it can be classified as no category in GHS (green arrows on Fig. 8a).
• If the ingredient is classified as a severe irritant (MSc ≥9) with a coagulation score higher than zero in HET-CAM (red arrows on Fig. 8a), it presents moderate or severe irritant potential (GHS category 2).
• If the ingredient has irritant potential (severe, moderate or mild) in the HET-CAM assay (MSc ≥9 or 1 ≤MSc ≤8.9) (orange and blue arrows in Fig. 8a, respectively), it cannot be classified accurately as Slight, Mild, Moderate or Severe irritant. Depending on the aim
analysis, it should also be submitted to BCOP assay and to histo- pathological analysis, such as if the focus is on whether the mecha- nism of action and the exact classification of the substance are needed.
BCOP provides information about mechanisms of toxicity such as opacification and especially the permeability effect which is important for surfactants. We then suggest the following analysis of the results for BCOP strategy (Fig. 8b):
•If the ingredient presents IVIS ≤3, it is classified as non-irritant in BCOP assay (green arrows), and can be classified as no category in GHS.
•If the ingredient is classified as a severe irritant BCOP (IVIS >55), the substance can be classified as GHS category 1 or as a severe/corro- sive irritant (red arrows).
•If the ingredient presents 3 <IVIS ≤55 in the BCOP assay (no pre- diction can be made) (blue arrows on Fig. 8b), it should be submitted to histopathological evaluation of the cornea) to obtain the final classification, as indicated in the IATA 263 (OECD, 2017a) and BCOP assay (OECD TG 437, 2020).
Thus, our strategies involving both cheap and simple HET-CAM assay and also BCOP assay combined with histopathological analysis can contribute to the development of cosmetic products containing solutions or mixtures of surfactants (i.e., shampoos), since they can eliminate surfactant solutions with high eye irritation potential (moderate and
severe eye irritants), i.e. BCOP IVIS >55 and HET-CAM (MSc ≥9) with a coagulation score higher than zero.
In BCOP assay, only IVIS >55 or < 3 eliminates further time- consuming histopathological evaluation. HET-CAM MSc ≥ 9 or 1 ≤ MSc ≤8.9 is not a standalone result depending on the focus of analysis and consequently should be submitted to a follow up study such as BCOP and histopathological analysis, whether the exact classification of the substance is needed.
Histopathological evaluations are usually performed in specialized laboratories and it takes several days to obtain the histological slices.
However, histopathological evaluation can be performed if the focus is on whether the mechanism of action and the exact classification of the substance are needed.
Finally, according to IATA, the BCOP test is not considered valid as a full replacement of the Draize test, since many surfactants fall into the
“no prediction can be made” interval (3 <IVIS ≤55). Thus, our low-cost strategy involving HET-CAM can also be performed and can eliminate further time-consuming histopathological analysis when MSc ≥9 with a coagulation score higher than zero or MSc ≤0.9, especially to eliminate surfactant solutions with high eye irritation potential (moderate and severe eye irritants).
5. Conclusion
The final classification obtained in this study was based on an approach where HET-CAM, BCOP and cornea histopathological analysis were employed. HET-CAM is a cheap, fast and good assay to identify Fig. 8. Representative scheme of the proposed strategies approach to the evaluation of the ocular irritation potential of cosmetic ingredients a) HET-CAM and b) BCOP followed by histopathological analysis.