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.

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.

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.

The within- and between-laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: Results of validation study implemented in four participating laboratories.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within- and between-laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two-step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within-laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between-laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within- and between-laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.

Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25 µm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

Immunological characteristics of Hatano high-and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats have been genetically selected on the basis of their two-way active avoidance behavior, and have been shown to differ in other behavioral and hormonal parameters. Since close interconnections among the nervous, endocrine and immune systems have been well documented, these two strains might possess differences in aspects of immunological action. In Experiment 1, plasma levels of IgG, IgM, complement 3 (C3), classical pathway hemolytic complement (CH50) and beta(2)-microglobulin were compared between males of the two strains at 5 and 24 weeks of age. Plasma levels of IgG and CH50 were lower in LAA than HAA rats at 5 weeks of age, whereas those differences disappeared at 24 weeks of age. There were no differences between the two strains in plasma levels of IgM, C3 and beta(2)-microglobulin. In Experiment 2, antibody production to sheep red blood cells (SRBC) and mitogen-induced lymphocyte proliferation were compared between 12-week-old males of the two strains. Antibody responses in the PFC assay, plasma anti-SRBC-IgM levels and spleen weights were higher in LAA than HAA rats. LPS-induced lymphocyte proliferation was greater in LAA than HAA rats. It was concluded that HAA rats show earlier development of immunological development, but that antibody production and mitotic response of B lymphocytes may be more pronounced in adult LAA than HAA rats. The strain differences observed in the immunological response may indicate the usefulness of using Hatano rats in studies of behavioral-immunological relationships.

Differences in sperm motion between high- and low-shuttlebox avoidance rats (Hatano strains).

Sperm from the caudal epididymis of 2 inbred strains of Sprague-Dawley (SD) rats, selected on the basis of their high- or low-shuttlebox avoidance responses, were analyzed for motion characteristics by a computer-assisted sperm motion analysis (CASA) system. Sperm motion in high-avoidance animals (HAA) was characterized by high velocities, high amplitude of lateral head displacement (ALH), and low beat cross frequency (BCF). Conversely, sperm from low-avoidance animals (LAA) displayed low velocities, low ALH, and high BCF. These characteristics in sperm motion were not changed by washing. Furthermore, after treatment with alpha-chlorohydrin (aCH) as a male antifertility agent affecting rat epididymal sperm motion, sperm velocities in HAA rats were significantly reduced to levels similar to those in untreated LAA rats. However, ALH and BCF in HAA rats treated with aCH were different from those in untreated LAA rats. Sperm adenosine triphosphate (ATP) content was higher in HAA than in LAA rats, correlating with values of their sperm velocities. These data suggest there are apparent strain differences in sperm motion between HAA and LAA rats and that these differences are dependent on factors, including sperm energy production.

Strain differences in the spontaneous incidence of sperm morphological abnormalities in Hatano rats.

Two inbred strains of Hatano rats, which had been bred selectively on the basis for shuttlebox avoidance responses (high- and low-avoidance animals; HAA and LAA), were different in sperm motility. We have found that there are strain differences in the spontaneous incidence of sperm morphological abnormalities. The percentages of abnormalities were extraordinarily higher in the LAA rats, whose sperm motility was lesser. Since the high sperm abnormality was an exceptional case in rats, these rats may be useful models for sperm quality.

Effective induction of oral anaphylaxis to ovalbumin in mice sensitized by feeding of the antigen with aid of oil emulsion and salicylate.

It is important to evaluate the ability of novel proteins in food crops and products to elicit potentially harmful immunologic responses, including allergic hypersensitivity. We developed a novel mouse model of food allergy involving an oral challenge of a protein antigen after feeding of the antigen in combination with modulating factors often ingested in daily life, namely, dietary oil emulsion and salicylate. In the model, BALB/c mice were sensitized orally for three weeks with ovalbumin (OVA) in linoleic acid/lecithin emulsion, followed immediately by intraperitoneal injection of sodium salicylate. At the end of the sensitization, the incidence of mice positive for serum OVA-specific IgG1 but not IgE had significantly increased in the combined-sensitization group. After the 3-week sensitization, a single or double oral challenge with OVA effectively and significantly caused severe anaphylaxis, as compared with the groups sensitized with OVA in the emulsion or the vehicle alone. Moderate increase of plasma histamine and intestinal abnormality in histology was found only in the combined-sensitization group. Anaphylaxis symptoms in the sensitized mice were induced more by oral challenge than by intravenous challenge, suggesting a critical role for the mucosal system. This is the first model for successful induction of oral anaphylaxis in mice sensitized by feeding of food protein without adjuvant. It will be useful to elucidate the mechanism of food allergy and to detect modulating factors of oral allergy at sensitization using this model, which simulates real life conditions.

Effects of transmaternal exposure to genistein in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats are separated by breeding from Sprague-Dawley rats by high versus low rates of avoidance responses in a shuttle-box task. In addition, compared to HAA rats, LAA rats show lower running-wheel activity, later sexual maturation, 5-day estrous cycling, lower sperm motility, more pronounced immunological reactions, and are generally less reactive to stress. The present study was designed to compare the effects of transmaternal exposure to genistein on these characteristics between HAA and LAA rats. To this aim, litters from both strains were fostered onto Sprague-Dawley rats receiving genistein by gavage with 5 mg/animal/day from day 17 of pregnancy through day 21 of lactation. Inhibited growth after weaning and reduced uterine weight at weaning were observed in the LAA offspring reared by genistein-treated dams. IgM antibody production in response to sheep red blood cells was significantly decreased in the HAA offspring reared by genistein-treated dams. During restraint stress, the plasma concentration of corticosterone was significantly lower in the LAA offspring reared by genistein-treated dams. Strain-related differences were detected in shuttle-box avoidance performance, running-wheel activity, estrous cycling, and sperm motility. The results demonstrate that transmaternal exposure to genistein potentially affects the immunological and stress responses as well as the post-weaning growth of the offspring. It suggests that a comparative study using Hatano rats would be useful for studying the influence of endocrine active chemicals on the whole body systems.