The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Evaluation of the sensitization potential of volatile and semi-volatile organic compounds using the direct peptide reactivity assay
Tsuyoshi KawakamiKazuo IsamaYoshiaki IkarashiHideto Jinno
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2020 Volume 45 Issue 11 Pages 725-735

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Abstract

The purpose of this study was to evaluate the sensitization potential of 82 compounds classified as volatile and/or semi-volatile organic compounds using the direct peptide reactivity assay (DPRA), given that these chemical compounds have been detected frequently and at high concentrations in a national survey of Japanese indoor air pollution and other studies. The skin sensitization potential of 81 of these compounds was evaluable in our study; one compound co-eluted with cysteine peptide and was therefore not evaluable. Twenty-five of the evaluated compounds were classified as positive. Although all glycols and plasticizers detected frequently and at high concentrations in a national survey of Japanese indoor air pollution were negative, hexanal and nonanal, which are found in fragrances and building materials, tested positive. Monoethanolamine and 1,3-butanediol, which cause clinical contact dermatitis, and several compounds reported to have weak sensitization potential in animal studies, were classified as negative. Thus, it was considered that compounds with weak sensitization potential were evaluated as negative in the DPRA. Although the sensitization potential of the formaldehyde-releasing preservative bronopol has been attributed to the release of formaldehyde (a well-known contact allergen) by its degradation, its degradation products—bromonitromethane and 2-bromoethanol—were classified as positive, indicating that these degradation products also exhibit sensitization potential. The compounds that tested positive in this study should be comprehensively assessed through multiple toxicity and epidemiological studies.

INTRODUCTION

As most people spend a major portion of the day in indoor environments, indoor air is an environmental medium that is highly relevant to human health. The Japanese government has addressed the safety of indoor air guidance establishing indoor concentration standards for 13 chemical compounds (Ministry of Health, Labour and Welfare, 2019) and regulating two chemical compounds in the Building Standards Law (Ministry of Land, Infrastructure, Transport and Tourism, 2003). However, during the decade since these indoor concentration standards were established, concerns have risen about indoor environmental pollution resulting from the use of alternative solvents and semi-volatile organic compounds (SVOCs). Against this backdrop, a committee tasked with investigating indoor air pollution (Committee on Sick Building Syndrome) was established in 2012 to discuss the revision of the indoor concentration standards and the addition of other compounds (Ministry of Health, Labour and Welfare, 2019). The Committee is in the process of conducting a nationwide survey to determine the status of indoor air pollution and releasing the results. Compounds of interest in relation to indoor air will be selected from the survey data and subjected to risk assessment before the indoor concentration standards are revised. However, there is little hazard information, particularly information on sensitization potential, regarding the risk assessment of these compounds.

Sensitization by chemical compounds falls under the categories of skin sensitization and respiratory sensitization. Many in vitro and in vivo assays have been established to evaluate the former, which is also known as a type IV allergy (delayed hypersensitivity reaction) (Aoyama, 2010). The latter generally appears as a type I allergy (immediate hypersensitivity). In the Japan Society for Occupational Health guidelines, respiratory sensitizers are defined as compounds that cause allergic respiratory disease (rhinitis, asthma, hypersensitivity pneumonitis, eosinophilic pneumonia, and other diseases with allergic involvement). These are categorized as compounds with confirmed sensitization potential in humans (group 1), compounds thought to have sensitization potential in humans (group 2), and compounds that may have sensitization potential in humans according to animal studies (group 3). Assessment criteria are defined for each group (Japan Society for Occupational Health, 2019). However, no in vivo or in vitro assay has been established for assessing respiratory sensitization potential (Aoyama, 2010; Mekenyan et al., 2014). Accordingly, very few chemical compounds have proven to be respiratory sensitizers (Japan Society for Occupational Health, 2019). Hence, this study sought to use the direct peptide reactivity assay (DPRA) to examine the skin sensitization potential of compounds classified as volatile organic compounds (VOCs) and/or SVOCs that were detected frequently and at high concentrations in a national survey of Japanese indoor air pollution and other investigations.

The DPRA is one of the non-animal methods to evaluate skin sensitization, and it is an in chemico skin sensitization assay adopted as an Organization for Economic Co-operation and Development testing guideline (OECD TG 442C) (OECD, 2015). It involves the evaluation of the binding of chemical compounds to proteins, which represents the initial step of the adverse outcome pathway (AOP) of skin sensitization. DPRA evaluates skin sensitization by quantifying the reactivity between test chemicals and two model synthetic peptides containing lysine and cysteine. In this investigation, we evaluated 82 compounds, including glycols, polycyclosiloxanes, fragrances, and preservatives.

MATERIALS AND METHODS

Reagents

The suppliers of the test compounds evaluated in this study are shown in Table 1. All compounds considered are VOCs or SVOCs, according to the definitions of the World Health Organization (WHO, 1989). Cinnamic aldehyde manufactured by Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) was used as the positive control. Sodium dihydrogen phosphate and disodium hydrogen phosphate used to make phosphate buffer solution were special-grade reagents manufactured by Kanto Chemical Co., Inc. (Tokyo, Japan)Ammonium acetate and aqueous ammonia used to make ammonium acetate buffer solution were manufactured by Nacalai Tesque, Inc. (Kyoto, Japan) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. Acetonitrile and trifluoroacetic acid (TFA) were high performance liquid chromatography (HPLC)-grade reagents manufactured by Sigma-Aldrich Japan (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd., respectively. Cysteine (Cys)-peptide and lysine (Lys)-peptide were purchased from Scrum Inc. (Tokyo, Japan). All water used for testing was passed through a Merk Millipore (Tokyo, Japan) Milli-Q Advantage A10 Ultrapure Water Purification System.

Table 1. CAS No, molecular weight, logKow, boiling point, classification, and suppliers of chemicals studied.

Phosphate buffer solution was prepared by mixing aqueous 100 mmol/L solutions of sodium dihydrogen phosphate and disodium hydrogen phosphate in a ratio of 18:82 (v/v) and adjusting the pH to 7.5 ± 0.05. Ammonium acetate buffer solution was prepared by dissolving 1.542 g of ammonium acetate in 200 mL of water and then using aqueous ammonia to adjust the pH to 10.2. To prepare the peptide solutions, Cys-peptide and Lys-peptide were dissolved in phosphate buffer solution and ammonium acetate buffer solution, forming Cys solution and Lys solution, respectively, with a final concentration of 0.667 mmol/L. Cinnamic aldehyde (positive control) and all test compounds were dissolved in acetonitrile to achieve a concentration of 100 mmol/L.

Methods

First, the test compound was mixed with a peptide that contains cysteine (Cys-peptide) or lysine (Lys-peptide) (Fig. 1) and allowed to react for 24 hr. Then, the amount of unbound peptide was determined, and the reactivity of the test compound was classified. When assessing the reaction between Cys-peptide and each of the test compounds, 200 μL of acetonitrile and 50 μL of the test compound solution were added to 750 μL of the Cys solution, mixed on a vortex mixer, and then allowed to stand at 25ºC for 24 ± 2 hr in the dark. The concentration of unreacted Cys-peptide was then determined with a HPLC/photodiode array detector (HPLC/PDA).

Fig. 1

Structures of peptides used in the study (top: Cys-peptide, bottom: Lys-peptide).

For assessments with Lys-peptide, 250 μL of each test compound solution was added to 750 μL of the Lys solution, mixed on a vortex mixer, and then analyzed using a method similar to that used for Cys-peptide. For all reactions with the peptides, a sample prepared by replacing the peptide solution with the corresponding buffer solution was used as a co-elution control, while a sample prepared by replacing the test compound solution with acetonitrile was used as a reference control. The co-elution control was used to check whether the retention time of each peptide overlapped with those of the test compounds, and the reference control was used to evaluate the stability of the peptides. A calibration curve was plotted for each peptide to confirm test validity.

In each of the tests, HPLC analysis was begun within 22 to 26 hr of mixing the initial sample and finished within 30 hr of starting the analysis. Peptide depletion by each test compound was calculated using the following formula:

For the DPRA, mean percent peptide depletion was calculated to evaluate the reactivity of the test compounds with the peptides and classified according to the models shown in Table 2 (OECD, 2015). The co-elution control was used to detect possible overlap in the elution times of the test compounds and each of the peptides. Overlap in the elution times of a peptide and test compound (i.e., co-elution) could hinder calculation of peptide depletion. However, if co-elution occurred for the Lys-peptide alone, classification was performed with the Cys-peptide 1:10 prediction model. If a depletion value was negative in the absence of co-elution, the mean was calculated with the depletion value considered to be zero. Retesting was performed when the peptide depletion value was near the positive criteria of the Cys-peptide 1:10/Lys-peptide 1:50 prediction model (3%-10%).

Table 2. Prediction models of DPRA.

HPLC/PDA conditions

A NexeraX2 System manufactured by Shimadzu Corporation (Kyoto, Japan) was used along with a Zorbax SB-C18 column (internal diameter 2.1 mm × length 100 mm × particle size 3.5 μm) manufactured by Agilent Technologies (Santa Clara, CA, USA). The column oven temperature was set at 30°C, and the autosampler rack temperature was set at 25°C. The flow rate was set at 0.35 mL/min, and the injection volume was set at 10 μL. The mobile phase consisted of Solution A, a 0.1% (w/v) aqueous solution of TFA, and Solution B, a 0.085% (w/v) solution of TFA in acetonitrile. The gradient was controlled by increasing the concentration of Solution B from an initial concentration of 10% to 25% over 10 min and then to 90% over the next 1 min. Solution B concentration was then held at 90% for 2 min, reduced to 10% in 0.5 min, and held at 10% for 7.5 min. The detection wavelengths were 220 nm and 258 nm, and peptide depletion was detected at 220 nm.

RESULTS AND DISCUSSION

Co-elution with peptides

HPLC chromatograms of two representative samples obtained by test-compound-free solution (reference control) are shown in Fig. 2. Of the 82 compounds tested in the study, 2-phenylethyl alcohol co-eluted with Cys-peptide, whereas diethylene glycol monoethyl ether acetate, isoeugenol, and methyldibromoglutaronitrile co-eluted with Lys-peptide. Co-elution control HPLC chromatograms of 2-phenylethyl alcohol and diethylene glycol monoethyl ether acetate are shown in Fig. 2. Of the co-eluting test compounds, diethylene glycol monoethyl ether acetate, isoeugenol, and methyldibromoglutaronitrile were evaluated using the Cys-peptide 1:10 prediction model. The compound 2-phenylethyl alcohol was not evaluated.

Fig. 2

HPLC chromatograms of reference control (left: Cys-peptide, right: Lys-peptide) and co-elution control (left: 2-phenylethyl alcohol, right: diethylene glycol monoethyl ether acetate). (reference control: peptide only, co-elution control: test substance only). (λ = 220 nm)

Test compound evaluation

The test results for all 82 compounds are shown in Table 3. The compound 2-phenylethyl alcohol co-eluted with Cys-peptide. Of the remaining 81 compounds, 25 were classified as positive, and the others were classified as negative. The DPRA has been shown to have over 80% correspondence with the mouse local lymph node assay (LLNA), which is widely used to test for sensitization potential. However, the DPRA often gives false negative results for compounds with weak sensitization potential and cannot be used for compounds that require metabolic activation (Tsujita and Ashikaga, 2014). In addition, the DPRA is unsuitable for accurately evaluating hydrophobic compounds because the peptide-test compound reactions are performed in aqueous solutions (Tsujita and Ashikaga, 2014). Our results must therefore be considered in light of these issues.

Table 3. Results of DPRA of chemicals studied.

The glycols, glycol ethers, plasticizers, polycyclosiloxanes, and related compounds detected frequently and at high concentrations in a national survey of Japanese indoor air pollution (Ministry of Health, Labour and Welfare, 2019) were all classified as negative. Twenty-three of the fragrances included in this study are mandated for labeling as allergens under European Union (EU) Cosmetics Regulations (EU, 2011). Of these 23 fragrances, 12 (from α-amylcinnamaldehyde to isoeugenol in Table 1) are considered “List A: Fragrance chemicals,” which according to existing knowledge, are most frequently reported and well-recognized consumer allergens, and seven of these 12 fragrances were classified as positive in this study. The other 11 fragrances are considered “List B: Fragrance chemicals,” which are less frequently reported and thus less documented as consumer allergens, and two of these were classified as positive. Thus, more of the List A fragrances widely recognized as allergens were classified as positive than the List B fragrances, although many compounds were classified as negative in our evaluation. Coumarin is an EU List A compound; however, it was classified as negative in our study and was also considered negative when the DPRA was developed (Gerberick et al., 2007). Both α-amylcinnnamaldehyde and α-hexylcinnamaldehyde were classified as weak sensitizers when the DPRA was developed (Gerberick et al., 2007); however, they were classified as negative in this study. Moreover, although benzyl benzoate and 3-(4-tert-butylphenyl) isobutyraldehyde are classified as weak sensitizers by LLNA, benzyl benzoate was considered negative and 3-(4-tert-butylphenyl) isobutyraldehyde weakly positive when the DPRA was developed (Gerberick et al., 2007); both were negative in this study.

The two compounds hexanal and nonanal not mandated for labeling under EU Cosmetics Regulations but detected in a national survey of Japanese indoor air pollution (Ministry of Health, Labour and Welfare, 2019), were classified as positive. Hexanal and nonanal are used industrially as fragrances and fragrance raw materials (Ministry of Health, Labour and Welfare, 2010, 2012) and are detected in indoor air when released from building materials derived from coniferous trees (Saito et al., 2010). Although a search for clinical investigation on the sensitization potential of these compounds revealed no relevant information, hexanal tested positive in LLNA (Patlewicz et al., 2004). Methyl jasmonate was classified as a weakly positive sensitizer but tested negative for sensitization potential in the guinea pig maximization test (GPMT) and human repeated insult patch test (Scognamiglio et al., 2012). When these 82 compounds were tested during the development of DPRA, some of the compounds found to be non-sensitizers in LLNA were classified as weakly positive (Gerberick et al., 2007). This may explain why a weakly positive result was obtained for methyl jasmonate in this study.

The compound 2-bromo-2-nitro-1,3-propanediol (bronopol) is used as a preservative in cosmetics and a variety of sanitary products (Kawakami et al., 2015). Although the sensitization potential of this preservative could not be evaluated using the GPMT (Noda et al., 2004), it was found to be a causative agent in clinical studies related to contact dermatitis (Frosch et al., 1990; Peters et al., 1983). Bronopol is a formaldehyde-releasing preservative, and it degrades into bromonitromethane and 2-bromoethanol as it releases formaldehyde (Fig. 3) (Kireche et al., 2011). Although the effects of formaldehyde have been implicated in sensitization by bronopol, bronopol itself might have sensitization potential because 15% of patients with a positive reaction to bronopol also have a positive reaction to formaldehyde (de Groot et al., 2010b). In a nuclear magnetic resonance study that used human cells, sensitization by bronopol induced reactivity different from that seen with formaldehyde and was augmented by 2-bromoethanol (Kireche et al., 2011). In this study, the DPRA was used to evaluate bronopol and its degradation products, bromonitromethane and 2-bromoethanol. These three compounds were classified as positive, indicating that not only bronopol but also its degradation products have sensitization potential. However, because bronopol releases formaldehyde and degrades more quickly with increasing temperature and pH (Kajimura et al., 2008), degradation effects at the time of testing must be factored in before classifying bronopol as positive. Bronidox and 1,3-dimethylol-5,5,-dimethylhydantoin (DMDM hydantoin) are also formaldehyde-releasing preservatives. DMDM hydantoin has been implicated in multiple cases of clinical allergic contact dermatitis (de Groot et al., 2010a, 2010b). In contrast, there are few reports of bronidox causing clinical sensitization (de Groot et al., 2010a, 2010b). Additionally, methyldibromoglutaronitrile has been reported to cause clinical sensitization (Erdmann et al., 2001). Bronidox, DMDM hydantoin, and methyldibromoglutaronitrile were all classified as positive in our study.

Fig. 3

Degradation process of 2-bromo-2-nitro-1,3-propanediol (bronopol) (Kireche et al., 2011).

Fumaric acid diesters and maleic acid diesters are used as preservatives and resin additives. The compound dimethyl fumarate is known to have strong sensitization potential, and related fumaric acid diesters and maleic acid diesters cross-react with dimethyl fumarate and have sensitization potential (Kawakami et al., 2012). In this study, the dimethyl, diethyl, and dibutyl esters of these compounds were classified as positive. However, the reported skin sensitizer diethylhexyl maleate (Imamura et al., 2017) and its isomer, diethylhexyl fumarate, were classified as negative. The DPRA is thought to be unsuitable for proper assessment of these compounds because they have a higher octanol/water partition coefficient than other compounds and therefore dissolve poorly in water. Recently, the amino acid derivative reactivity assay (ADRA) (OECD, 2019), which uses nucleophilic reagents containing cysteine or lysine bonded to a naphthalene ring, was adopted as OECD TG 442C. The ADRA can be used to evaluate test compounds at concentrations 20 to 100 times lower than those used in the DPRA and to evaluate compounds of hydrophobicity greater than what is evaluable with the DPRA. The ADRA should therefore be preferred when evaluating the hydrophobic compounds that could not be evaluated in this study.

Three aromatic primary amines were tested in this study. Of these, 2,4-daminotoluene was classified as positive; its sensitization assessment is in dispute because of differences in the interpretation of GPMT results (Chemical Evaluation and Research Institute, 2006a). Its structural isomer, 2,6-diaminotoluene, was classified as negative. The compound 4,4’-methylenedianiline tested positive in the GPMT (Chemical Evaluation and Research Institute, 2006b); however, it was classified as negative in our study, indicating that this may be a false negative result.

The three ethanolamines evaluated in this study were not found to have sensitization potential in animal studies and were classified as negative in this study. However, occupational contact dermatitis from monoethanolamine has been reported in metalworkers (machinists) (Lessmann et al., 2009). This is thought to result from frequent exposure of workers whose hands have a damaged skin barrier to monoethanolamine-containing lubricating oil (cutting fluids). Similarly, 1,3-butandiol, which was classified as negative in this study, has been associated with many cases of contact dermatitis from the use of cosmetics (Aizawa et al., 2014; Sugiura et al., 2001). Thus, these compounds probably have weak sensitization potential but cause many cases of sensitization in the clinic because their manner of usage entails frequent exposure. These compounds require comprehensive evaluation that factors in their exposure profiles and uses other assay procedures.

Respiratory sensitization potential of test compounds

No in vivo or in vitro assay has been established for assessing respiratory sensitization potential (Aoyama, 2010; Mekenyan et al., 2014). Lalko et al. used the DPRA to evaluate known respiratory sensitizers and compounds with skin sensitization potential, but without respiratory sensitization potential, and determined the ratio of Lys-peptide to Cys-peptide depletion (Lys:Cys ratio) (Lalko et al., 2012). They found that compounds with respiratory sensitization potential had a higher Lys:Cys ratio than compounds with only skin sensitization potential, noting that the Lys:Cys ratio of respiratory sensitizers is ≥ 0.2. This difference in the binding of DPRA test compounds to Cys- and Lys-peptides has been postulated to be a possible measure of respiratory sensitization potential (Mekenyan et al., 2014). However, it is not feasible to differentiate between skin and respiratory sensitizations with only the DPRA; hence, multiple test procedures should be used for evaluation of the AOPs of respiratory sensitization to the test compound in question (Dik et al., 2016).

Although about half of the compounds classified as positive in this study have Lys:Cys ratio exceeding 0.2, cinnamyl alcohol and 2-bromoethanol had borderline positive peptide depletion rates, and Lys-peptide depletion differed substantially between the first and second runs. This indicates that Lys:Cys ratios should not be used to evaluate the respiratory sensitization potential of weakly positive compounds. The compound dimethyl fumarate is implicated in many cases of contact dermatitis in Europe, causing not only skin symptoms but also sick building syndrome (Kawakami et al., 2012). It has a Lys:Cys ratio of 0.46. Therefore, respiratory sensitization potential must be comprehensively assessed to map AOPs through multiple toxicity and epidemiological investigations.

In conclusions, we used the DPRA to evaluate the skin sensitization potential of 82 volatile and/or semi-volatile compounds; 81 of these compounds were evaluable by the DPRA in our study. Twenty-five of the evaluated compounds were classified as positive. Although all glycols and plasticizers detected frequently and at high concentrations in a national survey of Japanese indoor air pollution were classified as negative through our evaluation, hexanal and nonanal, which are found in fragrances and building materials, tested positive. Monoethanolamine and 1,3-butandiol, which cause clinical contact dermatitis, and several compounds reported to have weak sensitization potential in animal studies, were classified as negative. This could be attributed to compounds with weak sensitization potential being evaluated as negative in the DPRA. Although the sensitization potential of the formaldehyde-releasing preservative bronopol has been attributed to the release of formaldehyde during its degradation to bromonitromethane and 2-bromoethanol, both of these products tested positive in our study, suggesting that these degradation products also have sensitization potential. The compounds that tested positive in this study should be comprehensively assessed in multiple toxicity and epidemiological studies.

ACKNOWLEDGMENTS

This study was supported by the grant H27-Kagaku-Ippan-009 from the Ministry of Health, Labor, and Welfare of Japan.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2020 The Japanese Society of Toxicology
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