Assessment of commercial polymers with and without reactive groups using amino acid derivative reactivity assay based on both molar concentration approach and gravimetric approach.

The amino acid derivative reactivity assay (ADRA), an alternative method for testing skin sensitization, has been established based on the molar concentration approach. However, the additional development of gravimetric concentration and fluorescence detection methods has expanded its range of application to mixtures, which cannot be evaluated using the conventional testing method, the direct peptide reactivity assay (DPRA). Although polymers are generally treated as mixtures, there have been no reports of actual polymer evaluations using alternative methods owing to their insolubility. Therefore, in this study, we evaluated skin sensitization potential of polymers, which is difficult to predict, using ADRA. As polymers have molecular weights ranging from several thousand to more than several tens of thousand Daltons, they are unlikely to cause skin sensitization due to their extremely low penetration into the skin, according to the 500-Da rule. However, if highly reactive functional groups remain at the ends or side chains of polymers, relatively low-molecular-weight polymer components may penetrate the skin to cause sensitization. Polymers can be roughly classified into three major types based on the features of their constituent monomers; we investigated the sensitization capacity of each type of polymer. Polymers with alert sensitization structures at their ends were classified as skin sensitizers, whereas those with no residual reactive groups were classified as nonsensitizers. Although polymers with a glycidyl group need to be evaluated carefully, we concluded that ADRA (0.5 mg/ml) is generally sufficient for polymer hazard assessment.

[In chemico skin sensitization alternative method: development of ADRA and listing to OECD test guideline].

Amino acid Derivative Reactivity Assay (ADRA) is an alternative method developed based on the principle of covalent bonding between sensitizer and proteins in the early stage of the mechanism of skin sensitization. The Direct Peptide Reactivity Assay (DPRA) with same principle previously listed in the OECD test guidelines (TG) have some problems such as precipitation of the test chemical in the reaction solution and co-elution of the peptide with the test chemical. While, instead of DPRA, the ADRA was developed using two chemically synthesized nucleophilic reagents-namely, NAC and NAL in which naphthalene rings with a high molar absorbance coefficient (MAC) in the ultraviolet range have been introduced to N-termini of the cysteine and lysine that can react with the test chemical. Therefore, in March 2016, we set up a validation team with the aim for adoption in the OECD TG, ADRA's validation tests were conducted. After reporting the results of validation study, holding a third-party evaluation meeting and two commenting rounds, ADRA was able to be adopted in the OECD TG in June 2019. In addition, since the introduction of naphthalene with a high MAC has made it possible to reduce the concentration, enabling the following items. 1) Decrease in the frequency of precipitation of the test chemicals in the reaction solution. 2) Decrease in the frequency of co-eluting of the nucleating reagent and the chemical. 3) Evaluation of chemicals with unknown molecular weight using the gravimetric approach. 4) High-sensitivity detection of nucleophilic reagents by the fluorescence method. 5) Evaluation of the mixture by a combination of the gravimetric approach and fluorescence detection.

Proportion of serum thyroid hormone concentrations within the reference ranges in athyreotic patients on levothyroxine monotherapy: a retrospective study.

BACKGROUND: In patients receiving thyroid-stimulating hormone (TSH) suppressive therapy with levothyroxine (LT4) after total thyroidectomy for thyroid cancer, thyroid function tests should be performed to adjust the LT4 dose. Specifically, serum TSH concentrations are commonly measured because TSH suppression is necessary according to thyroid cancer risk. The aim of the present study was to elucidate whether free thyroxine (FT4) or free triiodothyronine (FT3) indicates better for adjusting the dose in athyreotic patients on LT4 monotherapy after total thyroidectomy. METHODS: We retrospectively studied the compatibility of free thyroid hormone (FT4 and FT3) concentrations with reference ranges in athyreotic patients on LT4 monotherapy after total thyroidectomy. RESULTS: We identified 2210 consecutive patients from their medical records. Of these patients, 250 had both FT4 and FT3 concentrations in addition to TSH. Two hundred seven had serum TSH concentrations below the reference range (0.5-5.0 µIU/mL), while 43 had them within the reference range. In the 207 patients with TSH concentrations below the reference range, 61 patients (29.5%) had FT4 concentrations within the reference range (0.9-1.7 ng/dL) and 146 patients (70.5%) had FT4 concentrations above the reference range. In contrast, 10 patients (4.8%) had FT3 concentrations below the reference range (2.3-4.0 pg/mL) and 8 (3.9%) had FT3 concentrations above the reference range; 189 patients (91.3%) had concentrations within the reference range. Of the 43 patients with TSH concentrations within the reference range, 25 (58.1%) had FT4 concentrations within the reference range and 18 (41.9%) had FT4 concentrations above the reference range. While, 11 patients (25.6%) had FT3 concentrations below the reference range and one (2.3%) had FT3 concentrations above the reference range; hence, 31 patients (72.1%) had FT3 concentrations within the reference range. CONCLUSION: This study showed that measuring FT3 concentrations rather than FT4 concentrations as the subsequent parameter of thyroid function might be more useful for disease management in terms of the proportion of serum thyroid hormone concentrations within the reference ranges. Furthermore, FT3 measurement could be useful in providing more detailed treatments, including avoiding more aggressive TSH suppressive therapy and identifying the presence of low T3 syndrome in the background.

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.

Applicability of amino acid derivative reactivity assay (4 mM) for the prediction of skin sensitization by combining multiple alternative methods to evaluate key events.

The amino acid derivative reactivity assay (ADRA) is an alternative method for evaluating key event 1 (KE-1) in the skin sensitization mechanism included in OECD TG442C (OECD, 2021). Recently, we found that ADRA with a 4-mM test chemical solution had a higher accuracy than the original ADRA (1 mM). However, ADRA (4 mM) has yet to be evaluated using integrated approaches to testing and assessment (IATA), a combination of alternative methods for evaluating KE. In this study, the sensitization potency of three defined approaches (DAs) using ADRA (4 mM) as KE-1 was predicted and compared with those of two additional ADRAs or direct peptide reactivity assay (DPRA): (i) "2 out of 3" approach, (ii) "3 out of 3" approach, and (iii) integrated testing strategy (ITS). In the hazard identification of chemical sensitizers, the accuracy of human data and local lymph node assay (LLNA) remained almost unchanged among the three approaches evaluated. Potency classifications for sensitization were predicted with the LLNA and human data sets using ITS. The potency classifications for the sensitization potency prediction accuracy of LLNA data using any alternative method were almost unchanged, at approximately 70%, and those with ITS were not significantly different. When ITS was performed using DPRA, the prediction accuracy was approximately 73% for human data, which was similar to that of the LLNA data; however, the accuracy tended to increase for all ADRA methods. In particular, when ITS was performed using ADRA (4 mM), the prediction accuracy was approximately 78%, which proved to be a practical level.

The magnitude of increased Levothyroxine dose during pregnancy in patients on thyroid-stimulating hormone (TSH) suppression treatment after total thyroidectomy for papillary carcinoma.

The dose of L-T4 replacement for hypothyroidism often needs to be increased after pregnancy. In our institution, patients are instructed to double the dose 2 days a week after pregnancy. However, there is scarce evidence supporting the need for a dose increase after pregnancy in patients with preconception thyroid-stimulating hormone (TSH) suppression (TSH <0.3 µIU/mL). This study aimed to determine the need for a dose increase in L-T4 among women with a TSH-suppressive dose of L-T4 before pregnancy. In this retrospective observational study, between January 2008 and December 2018, we analyzed 166 pregnancies in 134 patients on TSH suppression treatment after total thyroidectomy for papillary carcinoma. Thyroid function tests were performed before and in the first trimester of pregnancy. The dose was adjusted and maintained during the first trimester of pregnancy in 76 pregnancies (group A) and 90 pregnancies (group B), respectively. The median serum TSH level was significantly lower in group A than that in group B (0.014 µIU/mL (IQR, 0.005-0.071) vs. 0.155 µIU/mL (IQR, 0.021-0.657), p < 0.001). TSH suppression could not be maintained after pregnancy in 15.8% and 38.9% of the pregnancies in groups A and B, respectively. Increasing the post-pregnancy dose by an average of 27.4% resulted in maintenance of TSH suppression after pregnancy in 84.2% of pregnancies. In conclusion, this study suggests that increasing the L-T4 dose after pregnancy may be appropriate in postoperative thyroid cancer patients whose serum TSH levels should be suppressed.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Hypothyroidism due to nephrotic syndrome: a notable clinical entity.

Nephrotic syndrome (NS) is characterized by massive urinary protein leakage and associated hypoproteinemia due to increased protein permeability caused by impaired renal glomerular connections. Although there have been several sporadic reports regarding the relationship between NS and thyroid dysfunction, a consensus has yet to be reached. The mechanism of hypothyroidism in NS is attributed to the loss of protein-bound thyroid hormones, such as thyroxine-binding globulin, transthyretin, and albumin, into the urine. Herein, we report four adults with hypothyroidism that developed or worsened due to the onset of NS. The patients' underlying thyroid status was post-total thyroidectomy with supplemental levothyroxine (L-T4) in two patients, hypothyroidism with supplemental L-T4 due to Hashimoto's disease in one patient, and Hashimoto's disease with normal thyroid function in one patient. Our results suggest that the presence of a reduced thyroid reserve may predispose patients to hypothyroidism in NS. We conclude that NS may cause or exacerbate hypothyroidism. In such cases, an NS assessment, including a urine test, is required.

Prevalence and Treatment Outcomes of Marine-Lenhart Syndrome in Japan.

INTRODUCTION: Marine-Lenhart syndrome (MLS) is now understood to be a combination of Graves' disease and autonomously functioning thyroid nodule(s) (AFTNs). The prevalence of the syndrome and suitable treatments for those living in iodine-sufficient areas are uncertain. OBJECTIVES: We aimed to investigate the prevalence, treatment, and prognosis of MLS in Japan, an iodine-sufficient area. METHODS: This study involved patients who visited our hospital between February 2005 and August 2019. Among patients with both thyrotoxicosis and thyroid nodule(s) larger than 10 mm, MLS and isolated AFTNs were diagnosed based on serum thyroid-stimulating hormone receptor antibody levels and scintigraphy using radioiodine or technetium-99m and thyroid uptake. RESULTS: Twenty-two patients were found to have MLS, compared to 372 with isolated AFTNs and 8,343 with Graves' disease, during the period. Therefore, the rate of MLS cases was 0.26% among all patients with Graves' disease (22/8,343). Treatments and outcomes were assessed for cases of MLS (n = 18) and isolated AFTNs (n = 269). Antithyroid drugs (ATDs) were withdrawn in 27.8% of cases in the MLS group and 10.3% in the isolated AFTN group. There was no significant difference in the clinical outcome after ATD withdrawal between the 2 groups. However, the rate of hypothyroidism after radioactive iodine (RAI) administration was significantly higher in the MLS group than in the isolated AFTN group (42.9 vs. 9.0%, p = 0.005) despite similar doses of RAI. CONCLUSIONS: The prevalence of MLS among patients with Graves' disease was 0.26% in Japan. RAI therapy induces hypothyroidism more frequently than in those with AFTNs probably because RAI is taken up in the surrounding Graves' tissues.

Cause Clarification of Cysteine Oxidation by Active Species Generated during the Oxidation Process of Cinnamaldehyde and Impact on an In Chemico Alternative Method for Skin Sensitization Using a Nucleophilic Reagent Containing Cysteine.

Aldehydes comprise a major portion of skin sensitizers because they can react with both cysteine and lysine. Moreover, cinnamaldehyde (CA) is a typical moderate sensitizer and is often used in an alternative test method for skin sensitization. The amino acid derivative reactivity assay (ADRA) is an in chemico test method that evaluates the reactivity of cysteine derivatives (N-(2-(1-naphthyl)acetyl)-l-cysteine, NAC) and lysine derivatives with the test chemicals and uses CA as a proficiency substance. We found that NAC depletion for CA was only 10-20% when CA was used directly from the reagent bottle, although it increased to almost 100% when stored after being aliquoted from the reagent bottle. It was also found that this was due to the air oxidation of NAC itself rather than the reaction of NAC with CA, indicating that this result simply shows an increase in apparent reactivity. Aldehydes are known to produce active species, such as radicals, during air oxidation. Therefore, we investigated whether radicals were generated under storage conditions using the radical scavenger OH-TEMPO. LC/MS/MS analysis revealed that CA and OH-TEMPO complexes were produced during the air oxidation of CA. In the results of five aldehydes, similar to CA, active species were not generated as significantly as CA. Collectively, during the evaluation of the aldehydes, it can be seen that careful measures need to be taken to prevent the aldehydes from oxidizing during storage, indicating that assessment without preventing air oxidation carries an increased risk of overestimation compared with the intrinsic skin sensitization potency.

Position paper from the Japan Thyroid Association task force on the management of low-risk papillary thyroid microcarcinoma (T1aN0M0) in adults.

The incidence of thyroid carcinoma has been increasing worldwide. This is interpreted as an increase in the incidental detection of papillary thyroid microcarcinomas (PTMCs). However, mortality has not changed, suggesting overdiagnosis and overtreatment. Prospective clinical trials of active surveillance for low-risk PTMC (T1aN0M0) have been conducted in two Japanese institutions since the 1990s. Based on the favorable outcomes of these trials, active surveillance has been gradually adopted worldwide. A task force on the management of PTMC in adults organized by the Japan Thyroid Association therefore conducted a systematic review and has produced the present position paper based on the scientific evidence concerning active surveillance. This paper indicates evidence for the increased incidence of PTMC, favorable surgical outcomes for low-risk PTMC, recommended criteria for diagnosis using fine needle aspiration cytology, and evaluation of lymph node metastasis (LNM), extrathyroidal extension (ETE) and distant metastasis. Active surveillance has also been reported with a low incidence of disease progression and no subsequent recurrence or adverse events on survival if conversion surgery was performed at a slightly advanced stage. Active surveillance is a safe and valid strategy for PTMC, because it might preserve physical quality of life and reduce 10-year medical costs. However, some points should be noted when performing active surveillance. Immediate surgery is needed for PTMC showing high-risk features, such as clinical LNM, ETE or distant metastasis. Active surveillance should be performed under an appropriate medical team and should be continued for life.

Amino acid derivative reactivity assay-organic solvent reaction system: A novel alternative test for skin sensitization capable of assessing highly hydrophobic substances.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing that focuses on protein binding. The ADRA is a skin sensitization test that solves problems associated with the direct peptide reactivity assay. However, when utilizing the ADRA to evaluate highly hydrophobic substances with octanol/water partition coefficients (logKow) of >6, the test substances may not dissolve in the reaction solution, which can prevent the accurate assessment of skin sensitization. Therefore, we developed the ADRA-organic solvent (ADRA-OS) reaction system, which is a novel skin sensitization test that enables the assessment of highly hydrophobic substances with a logKow of >6. We discovered that the organic solvent ratio, the triethylamine concentration, and the ethylenediaminetetraacetic acid disodium salt dihydrate concentration participate in reactions with the nucleophile N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and sensitizers that are used in ADRA and in stabilizing NAC. Thus, we determined the optimal reaction composition of the ADRA-OS according to L9 (33 ) orthogonal array experiments. Using this test, we assessed 14 types of highly hydrophobic substances. When we compared the results with ADRA, we found that ADRA-OS reaction system has high solubility for highly hydrophobic substances and that it has a high predictive capacity (sensitivity: 63%, specificity: 100%, accuracy: 79%). The implication of the results is that the novel ADRA-OS reaction system should provide a useful method for assessing the skin sensitization of highly hydrophobic substances with a logKow of >6.

Association between serum thyroid hormone balance and thyroid volume in patients treated with levothyroxine monotherapy for hypothyroidism.

Many previous studies including ours have reported that athyreotic patients on levothyroxine (LT4) have relatively low serum free triiodothyronine (FT3) levels, whereas patients with large goitrous diseases often have high serum FT3 levels. Here we investigated Hashimoto thyroiditis (HT) patients on LT4 to study the relationship between thyroid volume (TV) and thyroid hormone status in hypothyroid patients on LT4. We retrospectively studied 408 euthyroid HT patients treated with LT4 for hypothyroidism; divided them as per TV and compared serum levels of free thyroxine (FT4) and FT3 and the FT3/FT4 ratio in each patient group with those in euthyroid matched control group. We also evaluated the association between serum FT3 level and FT3/FT4 ratio and TV among HT patients on LT4. In patients with TV <15 mL, serum FT3 levels were significantly lower than those in controls. In patients with TV 15-80 mL, serum FT3 levels were equivalent to those in controls. In patients with TV ≥80 mL, the serum FT3 levels were significantly higher than those in controls. The serum FT3 level (r = 0.35, p < 0.01) and FT3/FT4 ratio (r = 0.42, p < 0.01) showed a positive correlation with TV. TVs in HT patients on LT4 caused differences in serum thyroid hormone balance, as increasing volume increases the serum FT3 level and FT3/FT4 ratio. Serum thyroid hormone balance in HT patients with smaller thyroids was similar to that in athyreotic patients. Mild thyrotropin suppression with LT4 is needed to achieve normal FT3 levels in such patients.

Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

Tumor Volume Kinetic Analyses Might Explain Excellent Prognoses in Young Patients with Papillary Thyroid Carcinoma.

INTRODUCTION: Young patients with papillary thyroid carcinoma (PTC) generally have excellent prognoses despite their often-advanced disease status. The reasons for this excellent prognosis are poorly understood. OBJECTIVE: To investigate the natural history of PTC in young patients, we compared the observed tumor volume-doubling rate (TV-DR) with the hypothetical tumor volume-doubling rate (hTV-DR) before presentation in young PTC patients. DR is an inverse of the doubling time and indicates the number of doublings that occur in a unit of time. A negative value indicates the number of times the volume is reduced by half per unit time. METHODS: We enrolled 20 patients with the following characteristics: age ≤19 years, diagnosed with PTC according to the cytology results between 2013 and 2018 and followed-up with periodical ultrasound examinations for ≥3 months before surgery for various reasons. Seventeen patients later underwent surgery confirming the diagnosis. We calculated TV-DRs using serial measurements of tumor diameters after presentation and hTV-DRs using tumor diameters and patients' age at presentation, assuming that a single cancer cell was present at the patient's birth and that the tumor grew at a constant rate. These values indicate the lowest growth rates necessary for a single cancer cell to achieve the full tumor size at presentation. RESULTS: Thirteen patients had positive TV-DRs (/year) ranging from 0.09 to 1.89, indicating tumor growth, and the remaining seven patients had negative values (-0.08 to -1.21), indicating regression. The median TV-DR was 0.29. The hTV-DRs (1.48-2.66, median 1.71) were significantly larger than the TV-DRs (p < 0.001), indicating much faster growth before presentation. CONCLUSIONS: These data suggest that deceleration of tumor growth had already occurred at presentation in the majority of the cases. This might explain why disease-specific survival is excellent despite frequent findings of advanced disease in young patients with PTC.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

Oxidation of a cysteine-derived nucleophilic reagent by dimethyl sulfoxide in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

Quality of Life in Patients with Low-Risk Papillary Thyroid Microcarcinoma: Active Surveillance Versus Immediate Surgery.

OBJECTIVE: This study aimed to compare the quality of life (QoL) and psychological issues of patients with papillary thyroid microcarcinoma (PMC) who were under active surveillance (AS) and those who underwent immediate surgery (OP). METHODS: This was a cross-sectional study conducted on 347 patients with low-risk PMC who were under AS (n = 298) or who underwent OP (n = 49). They were asked to complete two questionnaires (thyroid cancer-specific health-related QoL [THYCA-QoL] and the Hospital Anxiety and Depression Scale [HADS]). The results between the AS and OP groups were compared. RESULTS: The mean ages of patients in the AS and OP groups were 58.6±12.5 and 58.4±13.1 years (P =.94), respectively, and the male ratios were 34/298 (11%) and 2/49 (4.1%) (P =.14), respectively. The median follow-up periods from diagnosis in the AS and OP groups were 56.5 months (interquartile range [IQR], 32 to 88 months) and 84 months (IQR, 64 to 130 months) (P<.001), respectively. In the THYCA-QoL questionnaire, the OP group had more complaints about "voice" (P<.001), "psychological" (P =.025), "problems with scar" (P<.001), and "gained weight" (P =.047) than the AS group. Other scales of the THYCA-QoL were comparable in the two groups. In the HADS questionnaire, the AS group had significantly better anxiety (P =.020), depression (P =.027), and total scores (P =.014) than the OP group. CONCLUSION: PMC patients in the OP group had more complaints and were more anxious and depressed than the AS group. These findings suggest that AS is a reasonable alternative to surgery for patients with low-risk PMC from the point of view of QoL and psychology. ABBREVIATIONS: AS = active surveillance; CI = confidence interval; HADS = Hospital Anxiety and Depression Scale; LT4 = levothyroxine; OP = immediate surgery; PMC = papillary microcarcinoma; PTC = papillary thyroid carcinoma; QoL = quality of life; STAI = State-Trait Anxiety Inventory; THYCA-QoL = thyroid cancer-specific health-related quality of life; TSH = thyrotropin.

The amino acid derivative reactivity assay with fluorescence detection and its application to multi-constituent substances.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for the prediction of skin sensitization potential. Although co-elution of test chemicals and nucleophilic reagents during HPLC analysis is sometimes problematic when using the Direct Peptide Reactivity Assay (DPRA), it rarely occurs when using ADRA. Nevertheless, the application of either of these tests to multi-constituent substances requires nucleophilic reagents capable of selective detection. With this issue in mind, the authors developed an ADRA fluorescence detection method (ADRA-FL), which utilizes the natural fluorescence of ADRA nucleophilic reagents. In this study, we demonstrate the efficacy of ADRA-FL by testing 82 test chemicals used in the development of both DPRA and the conventional ADRA (ADRA-UV) as well as establish a threshold value for distinguishing sensitizers and non-sensitizers. Our results show that not only are depletion values obtained using ADRA-FL virtually identical to those obtained using ADRA-UV, the threshold value for either test is 4.9%. Additionally, in order to demonstrate the applicability of ADRA-FL to multi-constituent substances, we prepared test samples that consisted of a set of 10 non-sensitizers combined with one of 10 different sensitizers and tested each using ADRA-FL. The test results were concordant with those obtained using ADRA-UV. Also, because ADRA-FL chromatograms showed a significant decrease in multiple peaks as well as extremely stable baselines, we conclude that ADRA-FL is a highly selective and highly accurate mans of quantifying nucleophilic reagents that is applicable to a wide variety of chemical substances.

Applicability of amino acid derivative reactivity assay for prediction of skin sensitization by combining multiple alternative methods to evaluate key events.

Amino acid derivative reactivity assay (ADRA) has previously been developed as an alternative method to direct peptide reactivity assay (DPRA) to evaluate key event 1 in skin sensitization mechanisms. However, when using alternative methods for skin sensitization, integrated approaches to testing and assessment (IATA) that combine the results of multiple tests evaluating different key events are generally required. To verify whether ADRA can be used in IATA, we replaced DPRA with ADRA in five IATA methods combining DPRA, KeratinoSens, and h-CLAT: (i) the "2 out of 3" approach, (ii) the "3 out of 3" approach, (iii) sequential testing strategy (STS), (iv) integrated testing strategy by scoring approach (ITS-SA), and (v) the "ITS by two methods approach" (ITS-2MA). The prediction accuracy of the "2 out of 3" approach using ADRA (1 mM) and ADRA (0.5 mg/mL) was 90.0% and 91.1%, respectively, for human data, and was very similar to that obtained using DPRA (91.1%). The "3 out of 3" approach also showed good predictability (83.2%) using either ADRA (1 mM) or ADRA (0.5 mg/mL) compared to DPRA. Regarding the accuracy of the prediction of sensitization intensity for the human data by the third classification, prediction accuracy using ADRA was almost the same as STS, ITS-SA, or ITS-2MA using DPRA. As a result, this study showed that ADRA can be used as a test method for key event 1 in the evaluation of skin sensitization by combining multiple alternative methods.