Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

In vitro RHE skin sensitisation assays: Applicability to challenging substances.

In the last 20 years, alternative approaches to the identification of skin sensitisation hazards have been at the forefront of the 3Rs and have helped refine the validation and acceptance processes. However, experience with the local lymph node assay showed that, post-validation, challenges still occurred, particularly when a wider diversity of chemical substances was addressed, a situation which will arise with validated in vitro alternatives. In the present work, a range of substances potentially challenging to assess in current nonanimal OECD test guidelines were evaluated in several of the emerging in vitro alternatives. Twelve such substances (of which just over half were known skin sensitisers) were assessed in 4 assays, all based on reconstructed human epidermis (RHE) models. For hazard identification, the overall predictive accuracy ranged around 70% for three assays, although for one (SensCeeTox), it fell below 50% when human data was used as the benchmark. In most cases, sensitivity was high, such that sensitisation was overpredicted. As the substances were challenging to assess in other nonanimal methods, the results indicate that the 3D RHE models may be a useful tool for assessing skin sensitisation potentials without needing to revert to animal use.

Building scientific confidence in the development and evaluation of read-across.

Read-across is an alternative approach exploited to address information requirements for risk assessment and for regulatory programmes such as the European Union's REACH regulation. Whilst read-across approaches are accepted in principle, difficulties still remain in applying them consistently in practice. Recent work within Cefic LRI and ECETOC attempted to summarize the state-of-the-art and identify some of the barriers to broader acceptance of read-across approaches to overcome these. Acceptance is undoubtedly thwarted partly by the lack of a systematic framework to characterize the read-across justification and identify the uncertainties particularly for complex regulatory endpoints such as repeated-dose toxicity or prenatal developmental toxicity. Efforts are underway by the European Chemical's Agency (ECHA) to develop a Read-Across Assessment Framework (RAAF) and private sector experts have also considered the development of a similar framework. At the same time, mechanistic chemical categories are being proposed which are underpinned by Adverse Outcome Pathways (AOPs). Currently such frameworks are only focusing on discrete organic substances, though the AOP approach could conceivably be applied to evaluate more complex substances such as mixtures. Here we summarize the deliberations of the Cefic LRI read-across team in characterizing scientific confidence in the development and evaluation of read-across.

Increased pH and inorganic phosphate in temporal seizure foci demonstrated by [31P]MRS.

To investigate alterations of brain metabolism associated with temporal lobe epilepsy, [31P]MRS studies were performed on the anterotemporal lobes of patients with medically refractory complex partial seizures. Interictally, the pH was significantly more alkaline in the temporal lobe ipsilateral to the seizure focus (7.25 vs. 7.08, p less than 0.05), and the inorganic phosphorous concentration was greater on the side of the epileptogenic focus (1.9 vs. 1.1 mM, p less than 0.05). These changes in pH and inorganic phosphate may represent metabolic alterations secondary to seizures. Alternatively, because alkalosis enhances neural excitability and may enhance seizure activity, the increased pH of the seizure focus may provide insight into the pathophysiologic mechanism of epileptic seizures.

Alterations in brain phosphorus metabolite concentrations associated with areas of high signal intensity in white matter at MR imaging.

Areas of high signal intensity in white matter are identified on brain magnetic resonance (MR) imaging studies in 25%-50% of elderly subjects. The authors used phosphorus-31 MR spectroscopy to characterize the metabolic status of hemispheric white matter brain volumes in 30 elderly subjects with white matter areas of high signal intensity at MR imaging. Compared with white matter volumes with no or minimal areas of high intensity, white matter volumes with extensive areas of high intensity evidenced a 26% decrease in the adenosine triphosphate (ATP)/inorganic phosphate (Pi) ratio (P = .03) and a 21% decrease in the ATP concentration (P = .05), with the Pi level unchanged. A pilot P-31 spectroscopic imaging study in a subject with a large, coalescing white matter area of high signal intensity demonstrated large reductions in metabolite concentrations in the high-signal-intensity area. These results suggest that extensive white matter areas of high signal intensity indicate a process that affects white matter cellular energy metabolism.

Decreased phosphorus metabolite concentrations and alkalosis in chronic cerebral infarction.

A study was performed to determine quantitatively the alterations in phosphorus metabolite concentrations and pH in regions of the human brain damaged by chronic stroke. Image-guided phosphorus-31 magnetic resonance spectroscopy was performed on the brains of eight healthy subjects and six patients with cerebral infarction of more than 3 months duration. Phosphorus metabolite concentrations in infarcted regions were reduced 8%-67%. Significant decreases occurred in phosphomonoester (PME), phosphodiester (PDE), and adenosine triphosphate (ATP) concentrations, while inorganic phosphate (Pi) and phosphocreatine (PCr) concentrations showed smaller, nonsignificant decreases. The PCr/ATP ratio was significantly increased, while the ATP/Pi ratio was somewhat lower. The phospholipid ratio PDE/PME was also significantly increased, while the ratios of phospholipid (PME, PDE) to phosphate (PCR, Pi) metabolites were significantly decreased. The pH of the infarcted region indicated significantly more alkalinity than in the normal brain. The results suggest that chronic stroke is associated with significant changes in brain metabolite concentrations and pH that are different from those reported for other brain diseases.

Characterization of prostate cancer, benign prostatic hyperplasia and normal prostates using transrectal 31phosphorus magnetic resonance spectroscopy: a preliminary report.

We assessed the ability of 31phosphorus (31P) transrectal magnetic resonance spectroscopy to characterize normal human prostates as well as prostates with benign and malignant neoplasms. With a transrectal probe that we devised for surface coil spectroscopy we studied 15 individuals with normal (5), benign hyperplastic (4) and malignant (6) prostates. Digital rectal examination, transrectal ultrasonography and magnetic resonance imaging were used to aid in accurate positioning of the transrectal probe against the region of interest within the prostate. The major findings of the in vivo studies were that normal prostates had phosphocreatine-to-adenosine triphosphate (ATP) ratios of 1.2 +/- 0.2, phosphomonoester-to-beta-ATP ratios of 1.1 +/- 0.1 and phosphomonoester-to-phosphocreatine ratios of 0.9 +/- 0.1. Malignant prostates had phosphocreatine-to-beta-ATP ratios that were lower (0.7 +/- 0.1) than those of normal prostates (p less than 0.02) or prostates with benign hyperplasia (1.1 +/- 0.2, p less than 0.01). Malignant prostates had phosphomonoester-to-beta-ATP ratios (1.8 +/- 0.2) that were higher than that of normal prostates (p less than 0.02). Using the phosphomonoester-to-phosphocreatine ratio, it was possible to differentiate metabolically malignant (2.7 +/- 0.3) from normal prostates (p less than 0.001), with no overlap of individual ratios. The mean phosphomonoester-to-phosphocreatine ratio (1.5 +/- 0.5) of prostates with benign hyperplasia was midway between the normal and malignant ratios, and there was overlap between individual phosphomonoester-to-phosphocreatine ratios of benign prostatic hyperplasia glands with that of normal and malignant glands. To verify the in vivo results, we performed high resolution magnetic resonance spectroscopy on perchloric acid extracts of benign prostatic hyperplasia tissue obtained at operation and on a human prostatic cancer cell line DU145. The extract results confirmed the differences in metabolite ratios observed in vivo. We conclude that transrectal 31P magnetic resonance spectroscopy can characterize metabolic differences between the normal and malignant prostate.

P-31 spectroscopy study of response of superficial human tumors to therapy.

Studies were performed to characterize phosphorus-31 magnetic resonance (MR) spectra obtained from 10 superficial human tumors outside the brain and to determine whether P-31 MR spectroscopy could allow detection of a response to therapy before a change in tumor size was measured. The ratio of phosphomonoester to adenosine triphosphate peak intensities (PME/ATP) was unusually large in all tumors studied. The average PME/ATP in lymphomas (1.8 +/- 0.5) was greater than in nonlymphoma cancers (1.1 +/- 0.15). The average PME/ATP for all tumors studied (1.4 +/- 0.5) was much greater than that of underlying skeletal muscle (0.23 +/- .09). Eight of the tumors were studied before and after therapy. Responders were distinguished from nonresponders on the basis of changes in tumor size. PME/ATP decreased during therapy in three lymphomas that responded to therapy. In an adenocarcinoma and Ewing sarcoma that did not respond to therapy, PME/ATP increased. PME/ATP remained constant in two squamous cell carcinomas that responded to therapy and decreased in one squamous cell carcinoma that decreased in size by 40% but was classified as a nonresponder. Changes in PME/ATP did not always parallel changes in tumor size during therapy. In two patients, a decrease in PME/ATP preceded a decrease in tumor size. In four patients, PME/ATP increased transiently during periods when tumor size remained constant.

Alterations in brain phosphate metabolite concentrations in patients with human immunodeficiency virus infection.

Human immunodeficiency virus (HIV)-infected individuals often demonstrate neuropsychiatric impairment; however, it is unclear how brain metabolism may be altered in such patients. We used in vivo phosphorus 31 magnetic resonance spectroscopy to noninvasively assess brain energy and phospholipid metabolism by measuring brain concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), and inorganic phosphate (Pi), as well as phospholipid compounds and intracellular pH. In study 1, 17 HIV-seropositive men with varying degrees of neuropsychiatric impairment and six control subjects were studied. Localized spectra were obtained from a heterogeneous 5 x 5 x 5-cm volume of interest (VOI). Patients with HIV infection had a significantly lower ATP/Pi ratio and a trend for a lower PCr/Pi ratio than did the control group. In addition, the ATP/Pi and PCr/Pi ratios were both significantly negatively correlated with overall severity of neuropsychiatric impairment. In study 2, three HIV-seropositive men with neuropsychiatric impairment were compared with 11 HIV-seronegative men. Localized phosphorus 31 magnetic resonance spectra were obtained from two relatively homogeneous VOIs: (1) a predominantly white matter VOI, and (2) a predominantly subcortical gray matter VOI. The three HIV-infected patients demonstrated significantly decreased ATP and PCr concentrations in the white matter VOI. These results suggest that HIV infection of the brain may impair brain cellular oxidative metabolism and that the degree of metabolic compromise may be related to the severity of neuropsychiatric impairment.

Spin echo 31P spectroscopic imaging in the human brain.

Spectroscopic imaging of phosphorus metabolites in the human brain has been carried out with two data acquisition methods: by observation of the free induction decay (FID) signal and by a short spin echo sequence. The resultant spectral images and spatially resolved spectra are compared. Spin echo observation is found to provide spectra of superior quality, and by suitably selecting the sequence timing, no significant increase in T2 losses, as compared with the FID method, is encountered. 31P images with approximately 3.5 cm spatial resolution are obtained within times of 37 min at 2.0 T field strength.

P-31 MR spectroscopy of normal human brain and brain tumors.

Image-guided phosphorus-31 magnetic resonance (MR)-localized image-selected in vivo spectroscopy was performed on normal human brain and brain tumors. Peak area ratios, absolute molar concentrations of metabolites, and pH were determined. T1 values in normal brain were measured. The most important finding was that the metabolite concentrations detectable with MR spectroscopy in brain tumors were reduced from 20% to 70%. Phosphomonoesters, phosphodiesters, and phosphocreatine (PCr) showed the greatest decreases, while inorganic phosphate (Pi) showed the least change. The PCr-Pi ratio was significantly reduced in tumors. The pH of brain tumors (7.12 +/- 0.03) was more alkaline than that of normal brain (6.99 +/- 0.01). The authors conclude that the metabolite concentrations and pH in human brain tumors differ significantly from those in normal brain. These differences may be ultimately useful in characterizing tumors in man.

Comparison of 31P MRS and 1H MRI at 1.5 and 2.0 T.

The goals of this study were to compare 31P magnetic resonance spectroscopy (MRS) and 1H magnetic resonance imaging (MRI) of human subjects and phantoms at 1.5 and 2.0 T. The 31P signal-to-noise (S/N) ratios in phantom standards and in localized volumes in human brain and liver were compared at 1.5 and 2.0 T. In addition, T1 values for 31P resonances in human brain, 31P linewidths of metabolites in human brain and liver, 1H S/N in a phantom standard, and MR image quality in human head and body were compared at the two field strengths. The results of our study showed that at the higher strength field, (1) in vivo 31P MRS studies benefited from up to 32% improvement in S/N; (2) in vivo 31P MRS studies also benefited from increased spectral dispersion; (3) the quality of MR head images remained comparable; and (4) body images showed some decrease in image quality due to increased chemical shift, and flow and motion artifacts.

Clinical MRS studies of the brain.

Image-guided 31P and 1H magnetic resonance localized spectroscopy was performed on patients with brain tumors, temporal lobe epilepsy, chronic brain stroke, and deep white matter lesions. Absolute molar concentrations of metabolites, peak area ratios, and pH were obtained. The important findings were that 31P metabolite concentrations were significantly reduced in tumors, infarcts, and deep white matter lesions. Similarly, 1H metabolite intensities were reduced in chronic stroke. In the seizure foci of epilepsy patients, in tumors, and in chronic stroke, the pH was more alkaline than the normal pH. Peak area ratios were altered in tumors (reduction of phosphocreatine/inorganic phosphate (PCr/Pi) and in chronic stroke (large increases in Cr/NAA and Cho/NAA). Finally, the spectroscopic imaging technique offers a versatile alternative to the "single point" techniques, producing spectra or images of the spatial distribution of individual 31P metabolites.

Phosphorus-31 magnetic resonance spectroscopy in humans by spectroscopic imaging: localized spectroscopy and metabolite imaging.

In in vivo phosphorus magnetic resonance spectroscopy (MRS), spectroscopic imaging (SI) can be used as a flexible localization technique, producing spectra from multiple volumes in a single examination. Presented here are phosphorus SI studies of human organs in which a selective-volume SI reconstruction was used rather than the usual array-format SI reconstruction. A linear predictor technique was used to estimate the initial points of the free induction decay missing because of the delay needed for phase-encoding gradients, significantly reducing the baseline artifacts which commonly complicate interpretation of SI spectra. In studies of heart, brain, liver, and kidney, the performance of SI was found to compare favorably with that of ISIS. SI phosphorus metabolite intensity images from a brain tumor patient were obtained at 2 X 2-cm in-plane resolution (with "slice" thickness of roughly 16 cm, determined by coil sensitivity) in 34 min, demonstrating the feasibility of obtaining clinically useful metabolite images in clinically reasonable examination times.

Clinical magnetic resonance spectroscopy of brain, heart, liver, kidney, and cancer. A quantitative approach.

Clinical studies using 31P and 1H MRS with a whole body 2.0 T MRI/MRS system are described. In most cases, techniques to quantitate absolute molar concentrations of metabolites in various organs were used. In the brain, AIDS, chronic stroke, and white matter lesions were associated with alterations of brain 31P metabolites. Epilepsy was associated with increased pH in the seizure focus. In the heart, dilated cardiomyopathy was associated with increased PDE/ATP while PCr/ATP was unchanged. In the liver, alcoholic hepatitis and cirrhosis were associated with diminished hepatic ATP while alcoholic hepatitis had increased pH and cirrhosis had decreased pH. This allowed differentiation of normal liver, alcoholic hepatitis, and alcoholic cirrhosis without biopsy. In the prostate, malignancy was associated with increased PME/ATP and decreased PCr/ATP. The PME/PCr was greatly increased in malignant prostate with no overlap in normals. Other cancers outside the brain had increased PME and effective treatment was often associated with diminished PME. 1H MRS of the brain was performed using ISIS and outer volume suppression pulses for volume localization. Excellent high resolution 1H water-suppressed spectra were obtained at echo times as short as 30 ms, showing well resolved peaks for lactate, N-acetylaspartate, glutamate, choline, creatinine, and inositol. 1H MRS demonstrated that the uptake of ethanol by the brain was slower than the rise of ethanol in blood. 31P spectroscopic imaging of the brain with resolution of 2.25 x 2.25 x 2.5 cm produced metabolic images and high resolution spectra from desired regions of interest.(ABSTRACT TRUNCATED AT 250 WORDS)