Defining the reactivity of nanoparticles to peptides through direct peptide reactivity assay (DPRA) using a high pressure liquid chromatography system with a diode array detector

Abstract

The possibility of inducing skin sensitization reactions following exposure to various chemicals can lead to skin diseases, and the evaluation of skin sensitivity to such substances is very important. However, as animal tests for skin sensitization are prohibited, the OECD Test Guideline 442 C was designated as part of an alternative testing method. Therefore, in this study, the reactivity of cysteine and lysine peptides to nanoparticle substrates was identified through HPLC-DAD analysis according to the skin sensitization animal replacement test method specified in the OECD Test Guideline 442 C. In this study, all criteria for skin sensitization experiments specified in OECD Test Guideline 442 C were satisfied. As a result of analyzing the disappearance rates of cysteine and lysine peptides for the five types of nanoparticle substrates (TiO₂, CeO₂, Co₃O₄, NiO, and Fe₂O₃) using the established analytical method, all were identified as positive. Therefore, our findings suggest that basic data from this technique can contribute to skin sensitization studies by providing the depletion percentage of cysteine and lysine peptides for nanoparticle materials that have not yet been tested for skin sensitization.

Introduction

The possibility of inducing skin sensitization reactions to various chemical compounds that exist in the cosmetics and pharmaceutical industries can cause skin diseases; and thus, skin sensitization evaluations of various substances that are applied to the skin is very important [1, 2]. Allergic contact dermatitis (ACD) is a delayed type of hypersensitivity due to repeated exposure, and allergic reactions to various chemicals are known as potential causes of dermatitis and eczema. Approximately 20% of the world’s population has ACD [3, 4], and the generation of covalent conjugates of hapten to skin proteins plays an important role in the biological process of skin sensitization [5]. Repeated exposure to these chemicals reacts with proteins to form hapten-protein conjugates, which may activate immune system, which in turn is a major cause of ACD [6]. Previous studies have identified thousands of different chemicals as skin sensitizers, with approximately 4000 chemicals associated with ACD induction [7]. Therefore, identifying the skin sensitization properties of these various chemicals is needed to better understand their potential contributions to ACD.

The skin sensitization test method for ACD is carried out by the Buehler test, using guinea pigs, and in recent years, in accordance with the trend of prohibiting animal testing in cosmetics, an alternative animal test method, the local lymph node assay (LLNA), has been developed and used for evaluation. However, this method has the disadvantage of using a mouse [8, 9]. In addition, in 2011, the European Union (EU) prohibited animal testing for cosmetic evaluation,; thus, alternative test methods are needed for skin sensitization evaluations [10]. The following in silico, in chemico (in chemico skin sensitization: Direct Peptide Reactivity Assay (DPRA), Organization for Economic Co-operation and Development (OECD) test guideline 442 C), in vitro (in vitro skin sensitization: ARE-Nrf2 luciferase test method, OECD Test Guideline 442D, other test methods, such as in vitro skin sensitization human cell line activation test (h-CLAT) and OECD Test Guideline 442E) have been recently listed in the OECD Test Guidelines and proposed as animal test replacement methods [11]. Among these alternative test methods, the OECD Test Guideline 442 C was developed as an in vitro skin sensitization test for DPRA and approved as an internationally recognized effective in vitro skin sensitization test [12, 13]. In DPRA an HPLC was adopted for analyzing cysteine and lysine peptides, by which the reactivity of test chemicals with peptides would be predicted [14, 15].

Because of nanoparticles’ usefulness in human living, especially in medicine and engineering, the possibility of exposure to them has greatly been increased. However, toxicity studies on nanoparticles have not been paid much attention until now [16]. Therefore, in this study, the reactivity of several nanoparticles with two hapten-peptides was evaluated by using an High-performance liquid chromatography-diode array detector (HPLC-DAD) according to the OECD TG 442 C, an alternative test method for skin sensitization.

Materials and methods

Peptide preparation

The peptides used were purchased from Peptron (Daejeon, Korea) and used as received without further purification. The purities of cysteine peptide (Ac-RFAACAA) and lysine peptide (Ac-RFAAKAA) were > 97% and > 99%, respectively. The structures of the cysteine and lysine peptides used in this study are shown in Fig. 1.

Fig. 1.

The structure of cysteine peptide (a) and lysine (b) peptide

The preparation of proficiency substances

The five proficiency test substances and other materials were selected based on OECD Test Guideline 442 C (Table 1). The proficiency material was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Table 1.

Test substances for reference (OECD Test Guideline 442 C)

Proficiency substances	CAS. no	Physical state	DPRA prediction OECD TG 442 C	Range of cysteine depletion (%)	Range of lysine depletion (%)
Oxazolone	15646-46-5	Solid	Positive	60–80	10–55
Benzylideneacetone	122-57-6	Solid	Positive	80–100	0–7
Farnesal	19317-11-4	Liquid	Positive	15–55	0–25
1-Butanol	71-36-3	Liquid	Negative	0–7	0-5.5
Lactic Acid	50-21-5	Liquid	Negative	0–7	0-5.5

Chemical reagents

Distilled water (HPLC grade, > 99.9%), acetonitrile (HPLC grade, > 99.9%), trifluoroacetic acid (HPLC grade, > 99.9%), and acetic acid (HPLC grade, > 99.9%) were obtained from Fisher Scientific (Waltham, MA, USA). trifluoroacetic acid (TFA), ammonium acetate, and sodium phosphate monobasic sodium phosphate dibasic for the preparation of the buffer solutions were purchased from Sigma Aldrich (St. Louis, MO, USA).

Method of HPLC-DAD analysis conditions

HPLC-DAD analysis was performed according to the analysis conditions specified in OECD Test Guideline 442 C. Analysis was performed on a Shimadzu HPLC system diode array detector (220 nm) with an Agilent Zorbax SB-C18 3.5 μm 2.1 × 100 mm column (Palo Alto, CA, USA). The mobile phase consisted of (A) ultrapure water with 0.1% TFA, and (B) acetonitrile with 0.085% TFA. The gradient consisted of 10% B, and a flow rate of 0.2 mL/min. The gradient was then increased to 30% B for 10 min and then to 90% B in 1 min. After holding for 2 min, the gradient was changed to 10% B in 0.5 min and then held for another 6.5 min (Table 2).

Table 2.

The HPLC-DAD analysis condition of peptide

Parameters	Conditions
Analytical column	Zorbax SC-C18 (2.1 mm × 100 mm, 3.5 μm)
HPLC	Shimadzu HPLC system
Detector	Diode Array detector (220 nm)
Solvent	Solvent A : 0.1% TFA in water
	Solvent B : 0.085% TFA in acetonitrile
Mobile phase	Final time (min)	Solvent
		A (%)	B (%)
	0	90	10
	10	75	30
	11	10	90
	13	10	90
	13.5	90	10
	20	90	10
Flow rate	0.2 mL/min
Column oven temperature	30 ℃
Injection volume	10 µl
Run time	20 min

Preparation of test chemicals and a standard calibration curve

The solubility of test chemicals was tested in accordance with OECD TG 443 C (OECD, 2015), and the all test chemicals tested were dissolved in acetonitrile up to 100 mM. Therefore, acetonitrile was selected as the solvent for each test substance. According to OECD TG 442 C, the cysteine and lysine peptides were dissolved in phosphate buffer (pH 7.5) and ammonium acetate buffer (pH 10.2), respectively, at a concentration of 100 mM, and all buffer solutions were stored at 4 °C. Then, the cysteine peptides were serially diluted in 0.1 M phosphate buffer (pH 7.5) and the lysine peptide in 0.1 M ammonium acetate buffer (pH 10.2), to obtain a final concentration of 0.667 mM. Standard solutions of cysteine and lysine peptides at concentrations of 0.534, 0.267, 0.1335, 0.0667, 0.0334, and 0.0167 mM were prepared and serially diluted with each buffer.

Preparations of test reactions and control substances

Each control group was prepared according to the OECD Test Guideline 442 C, and each of the three independent controls was allowed to react at 25 ± 2.5 °C for 24 h and then injected into HPLC and analyzed three times. If a precipitate appeared during observation, the precipitate was centrifuged at a low speed to separate the precipitate. Each control group was prepared as follows:

Preparation of reference control A

To prepare reference control A, a cysteine peptide solution was prepared by adding 200 µL of acetonitrile to 750 µL of peptide solution and 50 µL of acetonitrile as a solvent. The lysine peptide solution was prepared by adding 750 µL of peptide solution to 250 µL of acetonitrile. Thus, the reference controls were prepared as follows:

− 0.667 mM cysteine peptide solution 750 µL + ACN 200 µL + ACN 50 µL.

− 0.667 mM lysine peptide solution 750 µL + ACN 250 µL.

Preparation of reference control C

Reference control (reference control) C was prepared by adding 200 µL of acetonitrile to 750 µL of the peptide solution and 50 µL of acetonitrile, as the solvent in which the test substance was dissolved. A solution of the lysine peptide was prepared by adding 250 µL of acetonitrile, as a solvent, to 750 µL of the peptide. The preparation methods were as follows:

− 0.667 mM cysteine peptide solution 750 µL + ACN + 200 µL ACN + 50 µL ACN (solvent to dissolve the test substance).

− 0.667 mM lysine peptide solution 750 µL + ACN 250 µL (Solvent to dissolve the test substance).

Preparation of positive control

For the preparation of the positive control, 100 mM cinnamaldehyde, as suggested in OECD TG 442 C, was used as a positive control, and 200 µL of acetonitrile was added to 750 µL of the cysteine peptide solution, to which 50 µL of 100 mM cinnamaldehyde was added. The solution was prepared by adding 250 µL of 100 mM cinnamaldehyde dissolved in acetonitrile to 750 µL of lysine peptide solution (final concentration 5 mM). The preparation method was as follows.

− 0.667 mM cysteine peptide solution 750 µL + ACN 200 µL + 100mM cinnamaldehyde 50 µL.

− 0.667 mM lysine peptide solution 750 µL + 100 mM cinnamaldehyde in ACN 250 µL.

Nanoparticle substances preparation and result analysis

Cysteine ​​and lysine peptide standard solutions were prepared at six concentrations (0.534–0.0167 mM), and a standard calibration curve was prepared. When the calibration curve of the peptide standard solution was analyzed by HPLC, the correlation coefficient (R²) was confirmed to be 0.99. In addition, it was confirmed that the peptide coefficients of variance of each control group were less than 15%, in accordance with OECD Test Guideline 442 C. The average loss rate of the three repetitions of the positive control group was within the acceptable range of 60.8 ~ 100% for the cysteine peptide and of 40.2 ~ 69% for the lysine peptide. The maximum standard deviations of the test substance and positive control were less than 14.9% for the cysteine peptide and less than 11.6% for the lysine peptide. The depletion percentage of the cysteine and lysine peptides for each test substance was determined as the average value of the repeated analysis, and the calculation of the depletion percentage for each test substance is shown below. The information on the five types of nanoparticle substances TiO₂ (Nanoamor, NM, USA), CeO₂ (American Element, CA, USA), Co₃O₄ (US-nano, TX, USA), NiO (Nanoamor, NM, USA), and Fe₂O₃ (Nanoamor, NM, USA) used in this study is specified in Table 3, and the particle sizes ranged from 10 to 100 nm.

Table 3.

The six types of manufacturing nanoparticle substances information

No.	Nanoparticles		CAS#	Primary size (nm)	M.W	Manufacturer
1	TiO2	Titanium (IV) oxide	13463-67-7	15–21	79.6	Nanoamor
2	CeO2	Cerium dioxide	1306-38-3	15–30	172.0	American Element
3	Co3O4	Cobalt (II, III) oxide	1308-06-1	50–80	240.8	US-nano
4	NiO	Nickel oxide	1313-99-1	10–20	74.7	Nanoamor
5	Fe2O3	Iron (III) oxide	1309-37-1	50–100	159.7	Nanoamor

$$\text{Percentage}\text{peptide}\text{depletion}\left(\%\right)=\left[1-\left(\frac{\text{Peptide}\text{peak}\text{area}\text{in}\text{replicate}\text{injection}}{\text{Mean}\text{peptide}\text{peak}\text{area}\text{in}\text{reference}\text{control}}\right)\right]\times 100$$

Definition of analysis results for test substances

For each test substance, the average cysteine peptide ​​and lysine peptide loss rates were evaluated as negative or positive according to the definition specified in OECD TG 442 C. If the value was positive with negative boundary values (borderline result, cysteine peptide ​​and lysine peptide loss rate of 3–10%), a retest was conducted.

Results

Cysteine peptide ​​and lysine peptide selectivity of the HPLC/DAD analysis

First, we confirmed whether the diluted buffer solution used in the experiment influenced the retention time of cysteine and lysine peptides under the analytical conditions specified in OECD TG 442 C. The dilution of cysteine ​​and lysine peptides in the pH 7.5 phosphate buffer solution and the pH 10.2 ammonium acetate buffer solution did not affect the retention time of the cysteine and lysine peptides, respectively. The cysteine ​​and lysine peptides were identified at 4.2 min (Fig. 2a) and 3.6 min (Fig. 2b), respectively, at a wavelength of 220 nm. In addition, the cysteine and lysine peptides could be detected at a lowest concentration of 0.0167 mM in the calibration curve sample presented in OECD TG 442 C, and sufficient sample was detected at the lowest concentration of the calibration curve (data not shown).

Fig. 2.

The chromatograms of HPLC-DAD for the lysine peptide and cystein peptide standard. Chromatograms of cysteine ​​peptide and lysine peptide in 0.534 mM sample. The cysteine peptide (a) and lysine peptide (b) standard were analyzed at 220 nm wavelength by dissolving them in pH 7.5 phosphate buffer and pH 10.2 ammonium acetate buffer

Confirmation cysteine ​​and lysine peptide linearity according to the concentrations of the calibration curve

In this study, six standard calibration curves were analyzed within the concentration range of 0.0167–0.534 mM, which is the calibration standard curve (with an R² > 0.99 for linearity) of the cysteine and lysine peptides that was specified in the DPRA analysis according to OECD TG 442 C. Both cysteine ​​and lysine peptides were serially diluted using a pH 7.5 phosphate buffer solution and a pH 10.2 ammonium acetate buffer solution, respectively, and each of the dilution buffers were specifically proposed in OECD TG 442 C. After preparation of the calibration curve, the samples were allowed to react at 25 ± 2.5 °C for 24 h. The calibration standard solution in the calculated quantitation ranges of both peptides was determined by plotting the corresponding peak areas determined using HPLC/UV. As a result of the linearity analysis, as shown below, the slope, intercept, and correlation coefficient (R²) were determined. The correlation coefficients (R²) of the cysteine peptide (Fig. 3a) and lysine peptide (Fig. 3b) were 0.9998 and 0.9996, respectively, meeting the criteria of R² > 0.99, as presented in OECD TG 442 C. Therefore, the concentration was determined by substituting the peak area of ​​all subsequent analyses into a calibration curve with confirmed linearity for each analysis batch.

Fig. 3.

Linear calibration curve response of cysteine peptide and lysine peptide. The concentration of the peptide was analyzed by diluting from 0.534, 0.267, 0.1335, 0.0667, 0.0334, and 0.0167 with each pH 7.5 phosphate buffer and pH 10.2 ammonium acetate buffer

Effectiveness evaluations of the reference and positive controls

The effectiveness of each reference and positive control specified in OECD TG 442 C was evaluated. Reference control A was used to verify the suitability of the HPLC analysis system, and reference control B was analyzed before and after the analysis sequence, to confirm the stability of the peptide over time. In addition, the reference control group C was analyzed before measurement of the test substance, to verify the stability of the peptide against the test solvent. As a result of the analysis, the concentrations of reference control A that were substituted for the calibration were 0.50 ± 0.05 mM (Table 4), and reference controls A and C showed coefficients of variation within 15.0% (Table 5). In addition, the standard deviation between repeated analyses of the test substances in the reference control group, including the positive control group, was 14.9% for the cysteine peptide ​​and 11.6% for the lysine peptide, which meets the criteria for each analysis item specified in OECD TG 442 C. Cinnamaldehyde specified in OECD TG 442 C was used as a positive control, and each sample was analyzed three times. In the analysis results, the average cysteine ​​peptide loss rate for the positive control group was 91.18%, and the average lysine peptide loss rate was 59.56%. The average depletion percentage of the positive control cinnamaldehyde in three repetitions was within the acceptable range for both the cysteine peptide (60.8 ~ 100%) and the lysine peptide (40.2 ~ 69%) (Table 6).

Table 4.

Evaluation of the effectiveness of the reference control A according to OECD TG 442 C

Sample	Peptide	Peak area	Measured concentration (mM)	Mean S.D (mM)	C.V (%)	Mean RE (%)
Reference control A (0.5 mM)	cysteine	4635325.0	0.4909	0.4952 0.0037	0.01	-0.95
		4696574.0	0.4974
		4694854.0	0.4972
	Lysine	3896979.0	0.5038	0.5041 0.0006	0.00	-0.81
		3896418.0	0.5037
		3904827.0	0.5048

 S.D standard deviation, C.V coefficient of variation, RE relative error

Table 5.

Evaluation of the effectiveness of the coefficient of variation for reference controls A and C according to OECD TG 442 C

Peptide	Sample	Peak area	Mean ± S.D	C.V (%)
Cysteine	Reference control A	4635325.0	4,707,687 42867.55	0.009
		4696574.0
		4694854.0
	Reference control C	4747310.0
		4720419.0
		4751637.0
Lysine	Reference control A	3896979.0	3,751,835 214200.2	0.057
		3896418.0
		3904827.0
	Reference control C	3859797.0
		3457116.0
		3495874.0

 S.D standard deviation, C.V coefficient of variation

Table 6.

Evaluation of the effectiveness of the positive control according to OECD TG 442 C

Sample	Peptide	Peak area	Peptide depletion(%)	Mean ± S.D (%)	C.V (%)
Positive control (cinnamaldehyde)	Cysteine	418308.0	91.17	91.18 0.01	0.00
		417738.0	91.19
		418036.0	91.18
	Lysine	1436062.0	60.16	59.56 0.67	0.01
		1453176.0	59.68
		1483641.0	58.84

 S.D standard deviation, C.V coefficient of variation

Analysis of proficiency substances and system properties

Within the concentration range of the peptides, with confirmed linearity, three positive substances (oxazolone, benzylideneacetone, and farnesal) and two negative proficiency substances (1-butanol and lactic acid) were selected from 10 proficiency substances using the analysis method specified in OECD TG 442 C, to verify the analysis method of this study system. As a result, the depletion percentages of the cysteine ​​and lysine peptides by the proficiency substance oxazolone were 63.04 and 21.29%, respectively, the depletion percentages by benzylideneacetone were 88.02 and 0.86%, respectively, and the depletion percentages by farnesal were 41.37 and 3.71%, respectively. In addition, the depletion percentages of the cysteine ​​and lysine peptides for 1-butanol were 1.01 and 0.62, respectively, and the depletion percentages for lactic acid were 2.31 and 1.14%, respectively (Table 7). Therefore, by comparing the results of this analysis with the aforementioned references, it was confirmed by HPLC-DAD chromatography that oxazolone, benzylideneacetone, and farnesal were positive substances and that 1-butanol and lactic acid were negative substances (Fig. 4), in agreement with the results specified in OECD TG 442 C.

Table 7.

Analysis of proficiency substances through system property

Test chemical	Peptide	Peak area	Peptide depletion (%)	Mean (%)	S.D.1 (%)	C.V.2 (%)	DPRA prediction (reference result)	DPRA prediction (reactivity class)
Oxazolone	Cysteine	1972508.0	62.82	63.04	0.81	0.01	Positive	Positive
		1913450.0	63.94
		1997604.0	62.35
	Lysine	4790755.0	21.71	21.29	1.65	0.08
		4928983.0	19.46
		4731952.0	22.68
Benzylideneacetone	Cysteine	666604.0	87.44	88.02	0.82	0.01	Positive	Positive
		605107.0	88.59
		548776.0	89.66
	Lysine	6179982.0	0.00	0.86	1.49	1.73
		6194825.0	0.00
		5962243.0	2.58
Farnesal	Cysteine	2902479.0	45.29	41.37	5.55	0.13	Positive	Positive
		3319067.0	37.44
		3368117.0	36.52
	Lysine	5858141.0	4.28	3.71	0.96	0.26
		5860224.0	4.25
		5961174.0	2.60
1-Butanol	Cysteine	4856807.0	1.16	1.01	0.20	0.19	Negative	Negative
		4875052.0	0.79
		4860567.0	1.09
	Lysine	3246793.0	1.87	0.62	1.08	1.73
		3395244.0	0.00
		3331190.0	0.00
Lactic acid	Cysteine	4777905.0	2.77	2.31	0.64	0.28	Negative	Negative
		4822654.0	1.86
		4719829.0	3.95
	Lysine	3273417.0	1.06	1.14	0.25	0.22
		3277715.0	0.93
		3261475.0	1.42

All values for the peak area are values obtained by subtracting the value of the peak area detected in the control group

 S.D standard deviation, C.V coefficient of variation

Fig. 4.

The analysis of proficiency substances through system property. The cysteine peptide and lysine peptide were reacted to 0.534 mM samples, respectively. HPLC-DAD analyses of the incubation mixture of test chemical with cysteine peptide (a) and lysine peptide (b) standard. Peptide reactions for proficiency substances were analyzed for each independent 3 sample after 24 h reaction at 25 ± 2.5 °C

Reactivity of cysteine ​​and lysine peptides with nanoparticles substrates

After determining the depletion rates of cysteine and lysine peptides on nanoparticle substrates using the analysis conditions specified in OECD TG 442 C, five types of nanoparticle substrates were determined as positive according to the definition specified in OECD TG 442 C. First, the depletion percentages of cysteine and lysine by TiO₂ were 24.13 and 2.23%, respectively, the depletion percentages of cysteine peptide ​​and lysine peptide by CeO₂ were 26.36 and 0.43, respectively, the depletion percentages by Co₃O₄ were 26.90 and 0.75%, respectively; the depletion percentages by NiO were 65.96 and 1.81%, respectively; and the depletion percentages peptide by Fe₂O₃ were 25.24 and 0.85%, respectively (Table 8).

Table 8.

Reactivity of cysteine peptide and lysine peptide to nanoparticles substrates

Test chemical	Peptide	Peak area	Peptide depletion(%)	Mean (%)	S.D.1 (%)	C.V.2 (%)	DPRA prediction
TiO2	cysteine	4190721.0	26.62	24.13	3.52	0.15	Positive
		4474838.0	21.64
		4473490.0	21.67
	Lysine	5062244.0	1.93	2.23	1.25	0.56
		5101886.0	1.16
		4976130.0	3.60
CeO2	cysteine	2869028.0	27.39	26.36	1.46	0.06	Positive
		2950360.0	25.34
		2947085.0	25.42
	Lysine	6007844.0	0	0.43	0.40	0.94
		5958659.0	0.49
		5940215.0	0.80
Co3O4	cysteine	287892.0	27.16	26.90	0.36	0.01	Positive
		2898650.0	26.64
		2908292.0	26.40
	Lysine	5981513.0	0.11	0.75	0.56	0.74
		5925311.0	1.05
		5922273.0	1.10
NiO	cysteine	2195052.0	64.71	65.96	1.14	0.02	Positive
		2102180.0	66.21
		2055388.0	66.96
	Lysine	6304748.0	3.09	1.81	1.25	0.69
		6392020.0	1.75
		6466866.0	0.60
Fe2O3	cysteine	3254728.0	17.63	25.24	10.76	0.43	Positive
		2653255.0	32.85
		3271097.0	17.22
	Lysine	6385403.0	1.85	0.85	0.88	1.04
		6474081.0	0.49
		6492604.0	0.20

All values for the peak area are values obtained by subtracting the value of the peak area detected in the control group

 S.D standard deviation, C.V coefficient of variation

Discussion

Since various substances to which skin is often exposed can cause skin sensitization and immunotoxicity, their evaluation is very important. Notably, ACD appears to be caused by a delayed type IV sensitization reaction mediated by T cells [17]. Previously, stimulation was mainly evaluated using the GPMT and Buehler methods in guinea pigs [18]. The development of alternative test methods to identify skin-sensitizing chemicals in animal models is still playing a key role in the cosmetics and pharmaceutical industries [19]. Several previous studies have reported the depletion rate of peptides for synthetic and novel chemicals through skin sensitization tests using the DPRA assay [20, 21]. However, few studies have investigated skin sensitization by nanoparticles, which frequently applied to the skin of modern people. Nanoparticles can be absorbed into the human body in various ways, and the skin is the primary absorption barrier. Accumulation of these nanoparticles may accompany not only cytotoxicity, but also diseases such as skin inflammation and skin cancer [22]. Therefore, this study evaluated the depletion of cysteine and lysine peptides by five types (TiO₂, CeO₂, Co₃O₄, NiO, Fe₂O₃) of nanoparticle substrates that have not been previously investigated for skin sensitization. The percentage depletion of cysteine and lysine peptides was evaluated by comparing the criteria specified in OECD TG 442 C with those reported in previous studies [23, 24]. According to a recent study, a series of platinum compounds, including hexachloroplatinates and tetrachloroplatinates, and two peptide models (cysteine and lysine) were investigated for depletion percent by OECD TG 442 C, and two palladium compounds with similar structures and cobalt chloride were additionally compared and tested [25]. Allergic contact dermatitis, occupational asthma, and allergic diseases have been associated with occupational environments that contain metals, metal oxides, and nanomaterials, and long-term exposure can result in immune side effects [26]. Therefore, in this study, the depletion percentage of cysteine and lysine peptides by five types of nanoparticles was investigated according to the OECD TG 442 C method. Our results revealed that the definition of depletion percentage of cysteine peptide and lysine peptides for the five nanoparticle substrates, TiO₂, CeO₂, Co₃O₄, NiO, and Fe₂O₃, was positive, which is expected to indicate skin sensitization upon penetration into the skin. In this study, an HPLC-DAD-based DPRA method was used as an established animal test alternative to predict skin sensitization. The rate of cysteine and lysine peptide loss in the presence of nanoparticles with unknown skin sensitization effects was identified. Therefore, this study investigated the skin sensitization of existing synthetic compounds, new compounds, and nanoparticle materials to which modern people may be exposed. Our results provide basic data that can contribute to skin sensitization research on nanoparticles during their development as medicines and cosmetics. In the process of peptide depletion by metals, various variables, such as oxidation or destruction of peptides, induction of peptide dimerization, and interference due to metals in HPLC analysis, may occur. Recently, in the case of substances that promote peptide oxidation, the possibility that the loss of peptides may be overestimated has been proposed[27, 28]. Therefore, to reduce the variables in the peptide loss evaluation process, the loss variables were verified using oxazolone, benzylideneacetone, farnesal, 1-butanol, and lactic acid, which are already known for their reactivity to peptides, as suggested by the OECD Test Guideline 442 C. This study proposes the possibility and usefulness of an alternative test method for animal experiments that can minimize the indiscriminate sacrifice of experimental animals, which has become a major issue in the recent R&D process, and evaluate skin sensitization.

Funding

There are no funding sources for this manuscript.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors have declared that there is no conflict of interest.