Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Quantitative polymerase chain reaction using an external control mRNA for determination of gene expression in a heterogeneous cell population.

Gene expression can be evaluated quantitatively by conventional RT-PCR or Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets. Therefore, we developed a new method for quantitative PCR using rat poly(A)+ RNA as an external control. We used this method to investigate cytokine gene expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control.

Development of a non-radioactive endpoint in a modified local lymph node assay.

A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for contact sensitization testing. Although the LLNA appears to be a little less sensitive than the most stringent of guinea pig assays, it provides a rapid, objective, quantitative and cost-effective method for screening strong contact sensitizers and has advantages with respect to animal welfare. However, a potential disadvantage is the need for the use of radioactive material. We have reported previously that an ex vivo assay based on similar principles to the original in vivo LLNA, but using a non-radioactive endopoint, was valid for the prediction of strong sensitizers. This ex vivo assay was not sensitive enough to allow prediction of moderately potent ones. In this study, we propose a new parameter, Corrected IL-2 Index (CII), for the prediction of moderate sensitizers. To obtain CII the IL-2 release in the supernatant of the cell culture is corrected for lymph node weight ratio and ratio of CD4-positive subset. We found that CII predicted the allergenicity of moderate sensitizers, including the ones recommended by the OECD in guideline 406, such as mercaptobenzothiazole and hexyl cinnamic aldehyde. The allergenicity of metal salts, such as potassium dichromate, ammonium tetrachloroplatinate and cobalt chloride, was also predicted by the CII. We conclude that the use of CII as an index significantly increases the sensitivity of the ex vivo method so that moderate sensitizers may also be detected.

UVA-induced immune suppression through an oxidative pathway.

Although ultraviolet B (UVB) irradiation induces local immune or systemic immune suppression, depending on the dose, the immune suppression by ultraviolet A (UVA) has not been fully investigated. In this study, we investigated the effect of UVA on the immune response in vitro and in vivo. The effect of UVA on the antigen-presenting function of epidermal cells was measured in terms of antigen-specific T cell proliferation. A murine epidermal cell suspension was exposed to UVA in vitro, pulsed with trinitrobenzenesulfonic acid, and cultured with T cells prepared from syngeneic mice previously sensitized with trinitrochlorobenzene. UVA (5-20 J per cm2) suppressed the antigen-presenting function of epidermal cells in a dose-dependent manner, accompanied with suppression of the expression of costimulatory molecules on Langerhans cells. In order to investigate the effect of an antioxidant on the immune suppression, an epidermal cell suspension was irradiated with UVA in the presence or absence of glutathione. The suppressions of antigen-presenting function and ICAM-1 expression were significantly prevented by glutathione in a dose-dependent manner. Further, the effect of UVA on the immune response at the induction phase of contact hypersensitivity was evaluated in terms of lymph node cell proliferation ex vivo. UVA irradiation suppressed the endogenous proliferation of lymph node cells in trinitrochlorobenzene-painted mice, and this suppression was significantly reversed by the application of glutathione to the skin during irradiation. These results suggest that UVA-induced immune suppression may be mediated by reactive oxygen species, at least in part.

Interlaboratory Validation of the in vitro Eye Irritation Tests for Cosmetic Ingredients. (4) Haemoglobin Denaturation Test.

Interlaboratory validation of the haemoglobin denaturation (HD) test on 38 cosmetic ingredients was conducted by five to eight participating laboratories. The HD test was evaluated as an alternative method to the Draize eye irritation test (Draize test) based on three indices of protein denaturation: the test substance concentration that induces 50% HD of the positive control (RDC(50)), a relative HD rate at 1% of the test substance (1%RDR) and a relative change in maximum absorption wavelength (1% lambdamax). The coefficients of variation associated with a positive HD test among the participating laboratories were within an acceptable range for practical application. The in vitro test results were in relatively good agreement with the Draize test. The correlation coefficient (r) between the in vivo maximal average Draize total score (MAS) and log (RDC(50)), 1%RDR and 1% lambdamax were -0.91, 0.67 and 0.79, respectively. The results revealed several limitations associated with the HD test: (1) the HD test cannot be applied to coloured test substances with a strong absorption, around 418nm; (2) water-insoluble test substances cannot be evaluated by RDC(50) or 1%RDR; (3) the HD test cannot be applied to strong acids that exceed the buffering capacity of a phosphate buffer solution; (4) the HD test cannot be used to determine the potential for eye irritation caused by factors other than protein denaturation, for example, polyoxyethylene octylphenylether (10 E.O.). Thus, the HD test alone is not appropriate for predicting eye irritation potential. Nevertheless, the good agreement between the HD test results and in vivo irritation scores as well as the ease of application suggest that this test may play an important role in a test system to determine eye irritation potential.

Comparison of the melanogenesis in human black and light brown melanocytes.

We examined how and to what extent the constitution of melanin and the expression, as well as the activity, of melanosomal proteins influence the production of melanin pigment by human black and light brown melanocytes, Mel (b) cells and Mel (l) cells, respectively. Melanin pigment in Mel (b) and Mel (l) cells consisted of a mixture of eumelanin and pheomelanin, and Mel (b) cells contained a larger amount. The signal intensity ratio of eumelanin to pheomelanin was similar in both cell types, though the two cell types differed in appearance. Tyrosinase activity and the amount of tyrosinase-related protein (TRP-1) of Mel (b) cells were higher than those of Mel (l) cells. Dopachrome tautomerase (DCT) activity and the amount of 6H5MICA were reduced in Mel (b) cells in comparison with Mel (l) cells. No significant difference in DHICA-converting activity or catechol-O-methyltransferase activity was found between Mel (b) and Mel (l) cells. There was no correlation between DHICA-converting activity and amount of TRP-1. These results suggest that the difference in the pigmentation of the two human melanocyte cell lines, Mel (b) and Mel (l), is derived from differences in the activity and expression of tyrosinase, TRP-1 and DCT, which affect the content and constitution of melanin polymers.

Antihypertensive and metabolic effects of doxazosin in hypertensive patients with concomitant non-insulin-dependent diabetes mellitus.

In this multicentre, non-randomized clinical study, 35 hypertensive patients with concomitant non-insulin-dependent diabetes mellitus were treated with the selective alpha 1 adrenoceptor blocker doxazosin mesilate for 24 weeks, either alone or together with existing antihypertensive therapy. The effects of the drug on blood pressure, glucose and lipid metabolism were examined. Daily administration of 0.5-8 mg doxazosin caused a significant reduction in both systolic and diastolic blood pressure decreased from 172 +/- 15/90 +/- 11 mmHg at the beginning, to 149 +/- 19/80 +/- 12 mmHg at the end of the trial. Heart rate was not affected. The plasma glucose level showed no change, whereas the haemoglobin A1c level decreased significantly from 7.4 +/- 1.2% to 7.1 +/- 1.2% (P < 0.01). Total and high-density lipoprotein cholesterol levels remained unchanged. The triglycerides level, however, decreased from 113 +/- 54 mg/dl at the beginning to 98 +/- 49 mg/dl at the end of the trial (P < 0.05). In a subgroup of patients, who showed hypercholesterolaemia ( > or = 220 mg/dl, n = 9) at the beginning, the total cholesterol level decreased from 252 +/- 33 mg/dl to 220 +/- 33 mg/dl (P < 0.05). Two patients complained of abdominal fullness or diarrhoea, but both were easily manageable. The results indicate that doxazosin is effective and safe in the treatment of hypertension in non-insulin-dependent diabetics and does not affect glucose metabolism. As for lipid metabolism in non-insulin-dependent diabetic patients, doxazosin seems to have a beneficial effect.

A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay.

The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Iad-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes.

A multi-centre study of the efficacy and safety of pravastatin in hypercholesterolaemic patients with non-insulin-dependent diabetes mellitus.

A multi-centre, non-randomized clinical study of 12-months' duration was performed in 112 patients with hyperlipidaemia associated with non-insulin-dependent diabetes mellitus to evaluate the clinical efficacy and tolerability of pravastatin and to assess its effect on glycaemic control. Patients were eligible for this trial if they fulfilled the following criteria; a high plasma total cholesterol level greater than 220 mg/dl associated with stable glycaemic control for at least 3-months' observation period. On entry, patients received 10 mg pravastatin per day (5 mg twice daily) for 12 months. Clinical efficacy was evaluated in 108 patients. The results showed that pravastatin induced a significant decrease in serum cholesterol level mainly with LDL-cholesterol. Total cholesterol levels were decreased significantly from 275 +/- 3 mg/dl to 222 +/- 4 mg/dl within 3 months of the start of treatment, and LDL-cholesterol decreased from 192 +/- 4 mg/dl to 137 +/- 4 mg/dl. After 12 months' treatment, total cholesterol and LDL-cholesterol levels were 216 +/- 4 mg/dl (p < 0.001) and 137 +/- 5 mg/dl (p < 0.001), respectively. HDL-cholesterol levels were increased from 51 +/- 2 mg/dl to 56 +/- 2 mg/dl at 3 months (p < 0.001) and 55 +/- 2 mg/dl at 12 months (p < 0.01). Triglyceride concentrations were also decreased from 173 +/- 11 mg/dl to 156 +/- 13 mg/dl at 3 months and 137 +/- 10 mg/dl at 12 months (p < 0.01). Fasting plasma glucose and glycated haemoglobin levels were not affected by pravastatin. Adverse events observed in 5 cases were always mild and reversible. These results indicate a clinical usefulness of pravastatin with high compliance in patients with hyperlipidaemia associated with non-insulin-dependent diabetes mellitus.

[Well-differentiated adenocarcinoma of the papilla of vater--report of a case].

Reported is the case of a 64-year-old female who was admitted to our clinic because of hyperamylasemia. Duodenal endoscopic examination revealed a tumor at the papilla of Vater. A biopsy specimens taken from the top of the tumor showed a well-differentiated adenocarcinoma. Under the diagnosis of cancer of the papilla of Vater, pancreaticoduodenectomy was performed. The first histological diagnosis was carcinoma in adenoma, but from the results of nucleus-gland ratio examined from 6 parts of the "adenoma", all of which were over 0.50, we finally concluded this case as de nove well-differentiated adenocarcinoma, and not carcinoma in adenoma. Thus, there is a possibility that past reports of adenoma or carcinoma in adenoma of the papilla of Vater might contain de novo well-differentiated adenocarcinoma.