[Examination of Analytical Method for Tris(2,3-dibromopropyl)phosphate and Bis(2,3-dibromopropyl)phosphate to Revise the Official Methods Based on "Act on the Control of Household Products Containing Harmful Substances"].

In Japan, the use of frame retardants [tris(2,3-dibromopropyl)phosphate: TDBPP and bis(2,3-dibromopropyl)phosphate: BDBPP] in several household textile products is banned under the "Act on the Control of Household Products Containing Harmful Substances." As the official analytical methods for testing these substances have not been revised for over 42 years, several issues such as the using of harmful reagents, have been pointed out. Therefore, we developed a new method to revise the official method in our previous study. In this study, the validity of the developed test method is evaluated at six laboratories using two types of textile samples spiked with TDBPP and BDBPP at three concentrations (4, 8, and 20 µg/g). TDBPP and BDBPP are extracted under reflux using methanol containing hydrochloric acid. TDBPP is analyzed using GC-MS, and BDBPP is also analyzed using GC-MS after methylation with trimethylsilyl diazomethane. Although the accuracy (70-120%), repeatability (<10%), and reproducibility (<15%) of a few samples, mainly low concentration samples, are out of range, overall, the concentration level of detection limits of TDBPP and BDBPP (8 and 10 µg/g) in official analytical methods are quantifiable with sufficient precision using the proposed method. Furthermore, harmful reagents are not used in this method. Thus, the method validated in this study is effective as a revised method for the testing of TDBPP and BDBPP in household textile products.

[GC-MS Analysis of Tris(1-aziridinyl)phosphine Oxide Flame Retardants in Textile Products].

Tris(1-aziridinyl)phosphine oxide (APO) used as flame retardant in textile products, such as curtains, carpets, and sleeping clothes, is prohibited in Japan under the "Act on the Control of Household Products Containing Harmful Substances." This study developed a GC-MS-based method to quantify APO more accurately and safely than the current official method. The APO in textile products was extracted with methanol, the extract was replaced with acetone instead of hexane as previously reported, and purified by florisil cartridge column. This cleanup method was instead of the harmful and carcinogenic dichloromethane used for open column to purify the sample in the official method, giving consideration to health of analysts. For accurate and sensitive quantification, deuterated compound, APO-d12, was used as a surrogate standard. The calibration curve displayed linearity within the 0.01-2.0 µg/mL range for APO. The detection limit for APO was 0.008 µg/g with S/N=5, which was 50 times more sensitive than the current detection limit of 0.4 µg/g, enabling the analysis of sufficiently low concentrations. The recoveries in non-treatment cloth and flame-retardant textiles were 73.5-126.6% and relative standard deviations were 3.3-24.6% when 2 µg APO was added to 0.5 g of samples, confirming that it can be analyzed satisfactorily. Thus, the developed method is applicable to textile products of various materials.

GC-MS Analysis of Primary Aromatic Amines Originated From Azo Dyes in Commercial Textile or Leather Products Using Helium Alternative Carrier Gases.

BACKGROUND: In recent years, due to the global shortage of helium gas, the development of gas chromatography (GC) analytical methods using alternatives to helium carrier gases is necessary. OBJECTIVE: The objective of this study was to examine the applicability of hydrogen and nitrogen as alternative carrier gases using the test method for azo compounds in the Act on Control of Household Products Containing Harmful Substances of Japan. METHOD: The gas chromatograph mass spectrometer (GC-MS) analytical method using hydrogen and nitrogen as alternative carrier gases was compared with a method using helium for 26 primary aromatic amines (PAAs) originated from azo dyes. RESULTS: When hydrogen and nitrogen were used as carrier gases under the same conditions used during analysis using helium (same column, gas flow rate, oven temperature conditions, etc.), sufficient peak separation of 26 PAAs was obtained. The sensitivities of the methods using helium and hydrogen were comparable, whereas the sensitivity was lower when nitrogen was used, with the detection limits ranging from 1/220 to 1/25. However, all carrier gases achieved quantification at concentrations below the standard value (30 µg/g) of the Act on Control of Household Products Containing Harmful Substances, and the results were in agreement with the standard value for the target product. CONCLUSIONS: Our results indicated that hydrogen or nitrogen can be used as alternative carrier gases to helium for GC-MS analysis of azo compounds producing specific aromatic amines. HIGHLIGHTS: Using hydrogen or nitrogen as an alternative carrier gas to helium, azo compounds could be quantified with excellent accuracy.

Development of a safer and improved analytical method for polycyclic aromatic hydrocarbons in creosote products.

Polycyclic aromatic hydrocarbons (PAHs) in creosote products used for wood preservation are regulated in Japan. Although the analytical method for this regulation has been stipulated by law, two main problems have been highlighted, namely the use of dichloromethane, a potential carcinogen, as a solvent and inadequate purification. Therefore, an analytical method to solve these problems was developed in this study. Actual creosote-treated wood samples were examined, and it was found that acetone could be used as an alternative solvent. Purification methods using centrifugation, silica gel cartridges, and strong anion exchange (SAX) cartridges were also developed. It was found that the SAX cartridges strongly retained PAHs, and an effective purification method was developed using this phenomenon, in which contaminants were removed by washing with diethyl ether/hexane (1/9 v/v), which could not be achieved with a silica gel cartridge. This strong retention was attributed to cation-π interactions. The analytical method developed in this study yielded good recoveries (81.4-113.0%) with low relative standard deviations (<6.8%), and the limit of quantification (0.02-0.29 µg/g) was significantly lower than the current creosote product regulation. Therefore, this method can safely and effectively extract and purify PAHs from creosote products.

Analysis and risk assessment of vinyl chloride emitted from aerosol products.

The objectives of this study were to develop a novel analytical method for quantifying vinyl chloride (VC) emitted from aerosol products, to provide analytical data on VC in aerosol products, and to evaluate consumer VC exposure by aerosol products. Our quantitative method involves absorbing VC into dimethyl sulfoxide and analyzing it using headspace gas chromatography/mass spectrometry. The correlation coefficients of the VC calibration curves were ≥ 0.9994 in the range of 0.16-80 µg/mL VC standard gases, which were prepared under either nitrogen or emission gases containing dimethyl ether or liquid petroleum gas. VC concentrations in these emission gases were calculated using a VC calibration curve from standard gases prepared under nitrogen; they were within ± 10% of the actual concentrations. We analyzed 39 household aerosol products; VC concentrations of 0.095, 0.098, and 0.28 µg/L were detected in three polyvinyl chloride spray paints. Consumer VC inhalation exposure level was estimated through an exposure scenario, and the hazard quotient was confirmed to be very low when comparing the exposure level with a cancer risk level of 10-5 for inhaled VC. These results suggest that the human health risk from VC in spray paint was low.

Species differences in activation of TRPA1 by resin additive-related chemicals relevant to indoor air quality.

Transient Receptor Potential Ankyrin 1 (TRPA1), which is expressed in the airways, has causative and exacerbating roles in respiratory diseases. TRPA1 is known as a target of sick building syndrome-related air pollutants, such as formaldehyde. Thus, an in vitro TRPA1 activation assay would be useful for predicting the potential risk of air pollution. In this study, we used human TRPA1 (hTRPA1)- and mouse TRPA1 (mTRPA1)-expressing cell lines to measure TRPA1 activation by the emerging indoor air pollutants 2-ethyl-1-hexanol (2-EH), a mixture of 2,2,4-trimethyl-1,3-pentanediol 1- and 3-monoisobutyrate (Texanol), and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). The results indicated that 2-EH activated both hTRPA1 and mTRPA1 in a concentration-dependent manner, whereas TXIB did not activate hTRPA1 or mTRPA1. Texanol also activated hTRPA1 in a concentration-dependent manner. In contrast, a bell-shaped concentration-dependent curve was observed for mouse TRPA1 activation by Texanol, indicating inhibitory effects at a higher concentration range, which was also reported for menthol, a typical TRPA1 modulator. To further elucidate the mechanism underlying the species difference in TRPA1 activation by Texanol, V875G and G878V mutations were introduced into hTRPA1 and mTRPA1, respectively, which were reported to be key mutations for the inhibitory effect of menthol. These mutations switched the inhibitory effects of Texanol; thus, hTRPA1/V875G, but not mTRPA1/G878V, was inhibited at higher concentrations of Texanol. These results indicate that Texanol shares an interaction site with menthol. Overall, these findings suggest that careful interpretation is necessary when extrapolating rodent TRPA1-dependent toxicological effects to humans, especially with respect to the risk assessment of indoor air pollutants.

Theoretical Validation of In Chemico Skin Sensitization Assay "ADRA" Using the Products Formed by Nucleophilic Reagents and Chemicals.

Amino acid derivative reactivity assay (ADRA) is an in chemico assay for assessing the skin sensitization potential of chemicals by evaluating the reactivity of nucleophilic reagents that mimic skin proteins. N-(2-(1-Naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), used as nucleophilic reagents, are small-molecule derivatives of two different amino acids, each with a naphthalene ring attached. The rate of decrease in the amount of NAC or NAL in the reaction solution is evaluated in this assay as an indicator of the test substance's skin sensitization ability. However, the products formed between the nucleophilic reagent and the test substance, which play an important role in vivo, are not directly identified. Therefore, six highly reactive chemicals, including the proficiency substances listed in the OECD Test Guidelines─squaric acid diethyl ester, 2-methyl-2H-isothiazol-3-one (MI), p-benzoquinone, palmitoyl chloride, diphenylcyclopropenone (DPCP), and imidazolidinyl urea (IU)─were used to determine each formed product. Samples were prepared according to the standard ADRA method, and the formed products were predicted on the basis of the reaction mechanism. Excluding DPCP, the estimated structures were validated using mass spectrometry and nuclear magnetic resonance spectrometry on the synthesized samples. In this manner, the products of each nucleophile were confirmed for all examined test substances. The estimated structure products were obtained through a series of reactions initiated by the nucleophilic attack of NAC's thiol group or NAL's amino group on the test substance's electron-deficient carbonyl carbon. However, contrary to expectations, disulfide-linked-type ring-opened products were detected in the case of MI, and products with free formaldehyde in solution were detected in the case of IU. In summary, all skin sensitizers tested herein reacted with NAC and/or NAL to give products. This supports the theoretical validity of ADRA, which provides an indirect evaluation of the formed products based on a decrease in nucleophilic reagents.

Determination of Carcinogenic Primary Aromatic Amines Contained as Impurities in Synthetic Organic Colorants.

BACKGROUND: Several primary aromatic amines (PAAs) have been designated carcinogenic or suspected of carcinogenicity. Several kinds of PAAs may occur either via the reduction of azo compounds or as impurities in azo colorants or other agents. OBJECTIVE: An analytical method was developed and applied to determine whether certain PAAs are present as impurities in synthetic organic colorants. METHOD: Target chemicals were analyzed by the ultrasound extraction of the synthetic organic colorant with a hydrochloric acid solution containing 20% methanol, followed by conversion from an acidic to alkaline solution, and then extraction using a diatomaceous earth column. RESULTS: We analyzed certain PAAs in 38 synthetic organic colorants, resulting in the detection of 2,4-dimethylaniline in four samples at 1.2 to 19 µg/g, o-toluidine in three samples at 1.0 to 3.4 µg/g, p-phenylazoaniline in two samples at 74 to 305 µg/g, and, in one sample each, 2,4,5-trimethylaniline (13 µg/g), 5-nitro-o-toluidine (12 µg/g), and 2-methyl-4-(2-tolylazo)aniline (13 µg/g). Nearly all PAAs were determined to be starting materials for colorant synthesis, although p-phenylazoaniline in Yellow No. 407 was apparently a byproduct formed during synthesis. For Red No. 225, in which high concentrations of p-phenylazoaniline were detected, additional samples were purchased from five companies, and p-phenylazoaniline was detected at concentrations of 88 to 370 µg/g in all samples. CONCLUSIONS: A method to analyze certain PAAs contained as impurities in synthetic organic colorants was developed, and the actual status of them in colorants was clarified. HIGHLIGHTS: The analytical method developed in this study for the determination of certain PAAs contained as impurities in synthetic organic colorants may be used to improve the safety of colorants.

[Investigation of Foreign Particles in Moderna COVID-19 Vaccine].

Particular batches of Moderna mRNA Coronavirus Disease 2019 (COVID-19) vaccine were recalled after foreign particles were found in some vaccine vials at the vaccination site in Japan in August 2021. We investigated the foreign particles at the request of the Ministry of Health, Labour and Welfare. Energy dispersive X-ray spectroscopy analysis suggested that the foreign particles found in the vials recalled from the vaccination sites were from stainless steel SUS 316L, which was in line with the findings of the root cause investigation by the manufacturer. The sizes of the observed particles ranged from <50 µm to 548 µm in the major axis. Similar foreign particles were also detected in 2 of the 5 vaccine vials of the same lot stored by the manufacturer, indicating that the foreign particles have already been administered to some people via vaccine. Observation of the vials of the same lot by digital microscope found smaller particles those were not detected by visual inspection, suggesting that more vials were affected. Contrarily, visual inspection and subvisible particulate matter test indicated no foreign particles in the vials of normal lots. Possible root cause and strategies to prevent such a deviation were discussed from technical and regulatory aspects.

Characterization of synthetic turf rubber granule infill in Japan: Volatile organic compounds.

There has been extensive studies on the composition of tires and industrial rubber. However, there is insufficient information on volatile organic compounds (VOCs) emitted from rubber granule products used to fill synthetic turf fields. In this study, we applied a passive sampling method for assessing the VOCs emitted from rubber granule products used for filling synthetic turf fields. We also performed a quantitative component analysis using a gas chromatography-mass spectrometer (GC-MS). The component analysis results of 46 rubber granule-based products showed the predominant presence of benzothiazole and methyl isobutyl ketone. The level of benzene, which the International Agency for Research on Cancer classifies as a substance with sufficient evidence for carcinogenicity to humans, was below the lower quantification limit in the products tested in this study. Our study included most of the rubber granule products used for synthetic turf fields in Japan (>95% of the products in the current domestic market of Japan). Therefore, we obtained a comprehensive overview of the VOCs emitted from the rubber granule-based products used in Japan's synthetic turf fields. Estimating the exposure to these airborne VOCs is essential to evaluate the adverse health effects of the VOCs emitted from these rubber granule-based products. Our sampling method and results can help provide key data for such risk assessment studies in the future.

Characterization of synthetic turf rubber granule infill in Japan: Rubber additives and related compounds.

We have conducted several studies with an overall goal of assessing the effects of rubber granules in synthetic turf on the health of athletes, other players, and children in Japan. As part of these studies, the investigation reported herein was aimed at analyzing the concentrations of rubber additives (vulcanization accelerators, antioxidants, and cross-linking agents) and related chemicals in 46 rubber infills prior to their use in synthetic turf fields in Japan. Of the 36 chemicals selected for targeted analysis, 26 were detected and quantified. Nontargeted analyses further identified and quantified 16 compounds derived from vulcanization accelerators, plasticizers, and other additives. The types and concentrations of the detected compounds varied both between products and within the same product; in the case of rubber infill products made from recycled rubber, this variation was caused by the different types of rubber products recycled as raw materials. Elution tests with four simulated biofluids (gastric juice, intestinal juice, saliva, and perspiration) revealed that the elution rates varied between compounds and were affected by the presence of coatings. Most compounds had low elution rates in all the simulated biofluids, with many at or below the limit of quantification. The data reported herein will be utilized in the risk characterization part of our subsequent study on the health risk assessment of rubber infill.

Characterization of synthetic turf rubber granule infill in Japan: Polyaromatic hydrocarbons and related compounds.

Although the health effects of artificial turf fillings have been investigated in Europe and the United States, the actual situation in Japan is unclear. To address this issue, the concentrations of 46 polyaromatic hydrocarbons (PAHs) and related compounds in rubber infills were analyzed prior to their use in synthetic turf fields in Japan. Based on information obtained from the sample suppliers, the investigated samples were divided into five categories: discarded tires, industrial rubber, combinations of these products or unidentified components (mixture/unknown), synthetic rubber specifically manufactured for synthetic turf, and special-purpose thermoplastic elastomers (TPEs). The industrial rubber samples were mixtures of styrene butadiene rubber, natural rubber, and ethylene propylene diene rubber (EPDM). The synthetic rubber samples consisted only of EPDM. A few or none of the PAHs were detected in the synthetic rubber and TPE samples. However, in the discarded tire and industrial rubber samples, benzo[a]pyrene, cyclopenta[cd]pyrene, and 30 other compounds were detected. A comparison between these two categories indicated that the discarded tire samples exhibited higher concentrations of the target compounds than the industrial rubber samples. This finding can be attributed to the presence of EPDM in almost all of the industrial rubber samples, which were not present in the discarded tire samples. The maximum PAH concentrations obtained in the present study were equivalent to or lower than the previously reported PAH concentrations. The total concentrations of the eight PAHs included in the European Chemical Agency (ECHA) assessment of health risks were lower in the present study than those reported by the ECHA. Furthermore, elution testing was performed with four simulated biofluids (gastric and intestinal juices, saliva, and perspiration). The actual elution amounts of all compounds were less than the limits. This report provides basic data for the risk assessment of PAHs in rubber infills.

Survival of SARS-CoV-2 and bovine coronavirus on common surfaces of living environments.

Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability.

Characterization of synthetic turf rubber granule infill in Japan: Total content and migration of metals.

We evaluated the total content of 28 metals in synthetic turf rubber granule infill and performed extraction tests using four types of simulated biofluids to assess the health effects of synthetic turf crumb rubbers used in Japan. The highest median metal concentration was obtained for Zn, with median concentrations above 100 µg/g, followed by Al, Fe, and Mn. The highest median Pb concentration was 19.9 µg/g. The metal concentrations of the samples were different depending on the origin/material. Among high-concentration metals, Al, Fe, and Mn were higher in ethylene propylene diene monomer rubber, and Zn was higher in tires. Significantly higher Sb and Sr concentrations were observed in other materials, including industrial rubber, synthetic rubber, and thermoplastic elastomer, compared with tires. However, significantly higher Sn, Co, Pb, and Cd concentrations were detected in tires compared with other materials. Metals with high concentrations independent of the origin/material were considered derived from materials added during the manufacturing process. To evaluate the bioaccessibility, extraction tests were conducted using simulated biofluids. In gastric fluid, many metals were detected in higher concentrations than in other biofluids, intestinal fluid, saliva, and sweat, and the extraction rate of most metals exceeded 10% in artificial gastric fluid. Because the amount of metals leached into the simulated biofluids was much lower than several standards on the amount of certain metals that have the potential to be extracted from the object if ingested, the risk related to the exposure to metals from synthetic turf rubber granule infill is considered low.

Effect of secondary particle size of nickel oxide nanoparticles on cytotoxicity in A549 cells.

The effect of nanoparticle type, shape, as well as primary and secondary particle size on toxicity remains poorly characterized. In this study, suspensions of nickel oxide (NiO) nanoparticles with the same primary particle size (< 50 nm) but different secondary particle sizes were prepared, and their cytotoxicity was investigated. A planetary ball mill wet nanopulverizer with zirconium milling balls of decreasing sizes (φ: 0.5, 0.1, and 0.05 mm) yielded NiO nanoparticles of decreasing mean particle size (310.4 ± 6.7, 172.0 ± 2.8, and 102.0 ± 0.5 nm). Stock solutions were diluted to various concentrations in 10% heat-inactivated fetal bovine serum containing minimum essential medium, and shown to have the same primary particle size, but different secondary particle sizes. Tests with A549 cells revealed that cytotoxicity increased with increasing secondary particle size: milling ball diameter φ 0.05 mm (IC50: 148 µg/mL) < φ 0.1 mm (IC50: 83.5 µg/mL) < φ 0.5 mm (IC50: 33.4 µg/mL). Uptake experiments indicated that the intracellular amount of Ni increased with increasing secondary particle size. In summary, the present findings show that differences in secondary particle size affected the cytotoxicity of NiO suspensions, which could be ascribed at least in part to differences in the amount of NiO taken up by the cells.

[Analysis of Flame Retardants Bis(2,3-dibromopropyl) phosphate and Tris(2,3-dibromopropyl) phosphate in Textile Products by GC-MS].

The use of flame retardants, namely bis(2,3-dibromopropyl) phosphate (BDBPP) and tris(2,3-dibromopropyl) phosphate (TDBPP), in textile products such as curtains, carpets and sleeping clothes is banned in Japan under the 'Act on the Control of Household Products Containing Harmful Substances'. Herein, we developed a GC-MS based method to quantify these compounds with greater accuracy and safety than the current official method. For accurate and sensitive quantification, deuterated compounds, BDBPP-d10 and TDBPP-d15, were used as surrogate standards. In consideration of the safety of the analyst, certain solvents and reagents used for the pretreatment that are carcinogenic or have a risk of explosion were replaced. For the extraction step, benzene was replaced by ethyl acetate, and for the methyl derivatization step, the reagent was changed from a self-prepared solution of diazomethane in ether to a solution of trimethylsilyl diazomethane in hexane, a safe and easy-to-use commercially available reagent. The calibration curves were liner in the range of 0.5-8.0 µg/mL for both methylated BDBPP (BDBPP-Me) and TDBPP. The detection limit was 0.05 µg/g for BDBPP-Me and 0.3 µg/g for TDBPP, which is sufficiently low compared to the current detection limits of 10 µg/g for BDBPP-Me and 8 µg/g for TDBPP. The recoveries in various curtain material were 66-108% and relative standard deviations were 1.2-10.2% when 5 µg BDBPP and TDBPP were added to 0.5 g of samples. Thus, the developed method is applicable to textile products of various materials.

Free formaldehyde in non-medical face masks purchased from the Japanese market since the COVID-19 outbreak.

Since the Coronavirus Disease 2019 (COVID-19) pandemic began, people have been wearing face masks for many hours every day. As these face masks are in contact with the skin, it is important to pay more attention to their quality and safety. This study examined the concentration of free formaldehyde in 90 non-medical face masks and related products (33 nonwoven, 30 woven cloth, 12 polyurethane, and 15 related products) because formaldehyde is a common contact allergen in textile products. For products consisting of mixed materials, each material was sampled, resulting in 103 samples for analysis. Free formaldehyde (34-239 µg/g) was found in three cloth masks, which consisted of cotton and polyester, with antibacterial and antiviral labeling. It was confirmed that the detected formaldehyde originated from the mask-finishing treatment by a hydrochloric acid extraction discrimination test. These masks may elicit contact dermatitis if the consumers have already been sensitized to formaldehyde. However, the risk of contact dermatitis caused by formaldehyde in masks may be considered low since the frequency of formaldehyde detection in masks in Japan is low.

Within- and between-laboratory reproducibility and predictive capacity of amino acid derivative reactivity assay (ADRA) using a 0.5 mg/mL test chemical solution: Results of the study for reproducibility confirmation implemented in five participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.