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.

Syrian Hamster Embryo (SHE) cell transformation assay with and without X-ray irradiation of feeder cells using Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG).

The SHE cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The need for an X-ray irradiated feeder cell layer necessitates the maintenance of an X-ray machine and the additional step to seed feeder cells prior to plating target cells. This laboratory has previously reported a method allowing target cells to be seeded in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer (Pant et al. [1,2,4]). In order to expand the data base for chemicals tested using this method, we describe in this paper the results obtained testing Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG) which are known to give positive responses in the standard SHE cell transformation assay. With freshly prepared conditioned medium (used within 2 weeks of preparation), there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In more than ten experiments the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without X-ray irradiated feeder cells. Compounds, DEHP and MNNG, were tested in the SHE cell transformation assay with and without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable between experiments performed using either method. These results demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and assisting the scoring of morphologically transformed colonies.

Determination of genetic toxicity and potential carcinogenicity in vitro--challenges post the Seventh Amendment to the European Cosmetics Directive.

Genetic toxicology and its role in the detection of carcinogens is currently undergoing a period of reappraisal. There is an increasing interest in developing alternatives to animal testing and the three R's of reduction, refinement and replacement are the basis for EU and national animal protection laws the Seventh Amendment to the EU Cosmetics Directive will ban the marketing of cosmetic/personal care products that contain ingredients that have been tested in animal models. Thus in vivo tests such as the bone marrow micronucleus test, which has a key role in current testing strategies for genotoxicity, will not be available for this class of products. The attrition rate for new, valuable and safe chemicals tested in an in vitro-only testing battery, using the in vitro tests currently established for genotoxicity screening, will greatly increase once this legislation is in place. In addition there has been an explosion of knowledge concerning the cellular and molecular events leading to carcinogenesis. This knowledge has not yet been fully factored into screening chemicals for properties that are not directly linked to mutation induction. Thus there is a pressing need for new, more accurate approaches to determine genotoxicity and carcinogenicity. However, a considerable challenge is presented for these new approaches to be universally accepted and new tests sufficiently validated by March 2009 when the animal testing and marketing bans associated with the Seventh Amendment are due to come into force. This commentary brings together ideas and approaches from several international workshops and meetings to consider these issues.

Metabonomics and the endocrine system.

Proton-NMR-based metabonomics offers a rare opportunity as a definitive screening technique for biofluids and tissue biopsies. The procedure is extraordinary in that it allows the 'complete biochemical picture' to be examined at one time and is able to detect subtle but repeatedly consistent disparities that may be occurring in different, and perhaps unrelated, biochemical pathways. Such metabolic responses to an initial perturbation in homeostasis may be followed over a sequential time-course to their eventual dissipation or consequent sequelae. The application of this technique is beginning slowly to filter into the area of endocrine research and has been used to examine long-term and diffuse physiological alterations that may occur following such events as anabolic steroid treatment of cattle and the exposure of endometrial cells to tamoxifen. Although only modest inroads have been made so far, this technique promises immense potential for future researches within the endocrine field.

The passage of trimethylamine across rat and human skin.

Trimethylamine is a volatile low molecular weight tertiary aliphatic amine that has known toxicity and the potential for human exposure from industrial and environmental sources is considerable. It is generally believed that absorption across the skin is an unimportant route of entry but there is little, if any, supporting evidence for this assumption. Passage across rat and human skin has been investigated employing excised skin circles in an in vitro diffusion cell apparatus. Trimethylamine was found to penetrate readily when applied to the epidermal surface of skin at three different dose levels (0.1, 1.0 and 10 mg per skin membrane 0.32 cm2). The apparent dermal flux was calculated as 3.40 +/- 1.60, 58.3 +/- 30.6 and 265.0 +/- 155.0 microg/cm2/h for rat and 0.98 +/- 0.75, 9.21 +/- 3.06 and 92.7 +/- 31.9 microg/cm2/h for human at the three dose levels, respectively. Both rat and human skin was able to act as a reservoir, with the trimethylamine not remaining in the stratum corneum but passing through. When presented to the underneath of rat and human skin circles, both [U-14C]-trimethylamine and [U-14C]-trimethylamine N-oxide were able to pass from the dermis to the epidermis. Small but detectable amounts of trimethylamine were oxidised to its N-oxide during passage through the skin.

In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study.

To obtain better insight into the robustness of in vitro percutaneous absorption methodology, the intra- and inter-laboratory variation in this type of study was investigated in 10 European laboratories. To this purpose, the in vitro absorption of three compounds through human skin (9 laboratories) and rat skin (1 laboratory) was determined. The test materials were benzoic acid, caffeine, and testosterone, representing a range of different physico-chemical properties. All laboratories performed their studies according to a detailed protocol in which all experimental details were described and each laboratory performed at least three independent experiments for each test chemical. All laboratories assigned the absorption of benzoic acid through human skin, the highest ranking of the three compounds (overall mean flux of 16.54+/-11.87 microg/cm(2)/h). The absorption of caffeine and testosterone through human skin was similar, having overall mean maximum absorption rates of 2.24+/-1.43 microg/cm(2)/h and 1.63+/-1.94 microg/cm(2)/h, respectively. In 7 out of 9 laboratories, the maximum absorption rates of caffeine were ranked higher than testosterone. No differences were observed between the mean absorption through human skin and the one rat study for benzoic acid and testosterone. For caffeine the maximum absorption rate and the total penetration through rat skin were clearly higher than the mean value for human skin. When evaluating all data, it appeared that no consistent relation existed between the diffusion cell type and the absorption of the test compounds. Skin thickness only slightly influenced the absorption of benzoic acid and caffeine. In contrast, the maximum absorption rate of testosterone was clearly higher in the laboratories using thin, dermatomed skin membranes. Testosterone is the most lipophilic compound and showed also a higher presence in the skin membrane after 24 h than the two other compounds. The results of this study indicate that the in vitro methodology for assessing skin absorption is relatively robust. A major effort was made to standardize the study performance, but, unlike in a formal validation study, not all variables were controlled. The variation observed may be largely attributed to human variability in dermal absorption and the skin source. For the most lipophilic compound, testosterone, skin thickness proved to be a critical variable.

DNA adducts, detected by 32P postlabelling, in human cholangiocarcinoma.

BACKGROUND: Reported mortality from intrahepatic cholangiocarcinoma (CCa) has risen steeply in the UK and other industrialised countries over the past 30 years, the cause of which has not been explained. DNA adduct formation is promutagenic and demonstrates exposure to a DNA damaging agent. It is a key step in chemically induced carcinogenesis. We hypothesise that the increase in CCa mortality is caused by a rise in a genotoxic environmental agent(s), causing cholangiocyte DNA damage. AIMS: To investigate and compare tumour and tumour adjacent CCa tissue, and non-cancer control bile duct tissue, for DNA adducts as a biomarker of genotoxin exposure. METHODS: DNA from 32 intrahepatic CCa patients (and in 28 cases DNA from adjacent non-tumour tissue) and from biliary ducts of seven non-cancer patients were investigated for the presence of DNA adducts using the nuclease P1 method of (32)P postlabelling. DNA adduct levels (number of adducts/10(8) nucleotides) were quantified. RESULTS: There was no significant difference in relative adduct labellings (RALs) between tumour adjacent DNA (median 8.6, range 1.2-51.6) and CCa DNA (7.2, 1.8-48.4). However, RALs were significantly higher in DNA from cancer patients (tumour adjacent and CCa DNA) compared with non-cancer patient DNA (2.9, 0.6-11.5; p=0.032, two tailed Mann-Whitney U test). Different adduct patterns were also seen in CCa compared with non-cancer patients. CONCLUSION: Quantitative and qualitative differences in adducts between cancer and non-cancer patients support the hypothesis that genotoxins may play a role in the development of intrahepatic CCa.

Cellular environment of metabolites and a metabonomic study of tamoxifen in endometrial cells using gradient high resolution magic angle spinning 1H NMR spectroscopy.

High resolution magic angle spinning (HRMAS) 1H NMR spectroscopy was used to metabolically characterise Ishikawa cells, a human cell line derived from endometrial adenocarcinoma. The spectra obtained had well-resolved resonances from the nucleotide derivatives of uridine and adenosine. Using a combination of diffusion- and relaxation-weighted spectroscopy, the cellular environment of key metabolites previously identified as related to cell growth was also investigated. As Ishikawa cells are hormone-responsive, the metabolic action of tamoxifen, a selective estrogen receptor modulator (SERM), was also investigated. Cells were exposed to 5, 1 and 0.1 microM tamoxifen. Using the statistical regression technique of prediction to latent structures by partial least squares, a predictive model was built modelling the metabolic profile of the cells against exposure to tamoxifen. These spectral changes were characterised by increased resonance intensities from ethanolamine (3.26 ppm), glucose (3.34-3.94 ppm), glutamate (2.14, 2.32 ppm), tyrosine (7.24 ppm), uridine (7.85 ppm) and adenosine (8.20 ppm), and a relative decrease in contributions from myo-inositol resonances (3.30, 3.62, 3.55 ppm). The nucleotide changes suggest that tamoxifen affects RNA transcription, while the changes in ethanolamine and myo-inositol concentrations are indicative of cell membrane turnover.

Mechanisms of hormonal carcinogenesis in the p53+/- hemizygous knockout mouse: studies with diethylstilbestrol.

The 2-year rodent bioassay has long had a central role in determining whether a compound is carcinogenic. It has recently been suggested that the use of 6-month studies in transgenic mice could reduce costs and animal numbers, without impairing the validity of cancer risk assessment. The p53+/- hemizygous knockout mouse model is phenotypically stable and develops tumors during the 6-month study period only in response to chemical and physical stimuli, showing a high concordance with genotoxic rodent carcinogens. We treated p53+/- mice and wild-type parent strain (C57BL/6J) animals with diethylstilbestrol (DES). 500 micromol/kg i.p. for 4 days. Following sacrifice, DNA was extracted from various tissues and adducts measured by a modified monophosphate version of the 32P-postlabelling assay. Major DES adducts were detected in the liver DNA of DES-treated wild-type mice at a level of 118.7+/-17.0 (mean +/- SD relative adduct level [RAL]/10(10) nucleotides) compared with 207.7+/-36.4 in p53+/- mice. No such adducts were detected in vehicle-treated animals. Total adduct levels, including endogenous I-compound adducts, in wild-type mice were 192.4+/-17.5 and 311.5+/-58.6 in p53+/- animals. These data support the hypothesis that deficient p53-dependent global genomic repair of DES adducts in p53+/- mice may result in the persistence of exogenous and endogenous DNA adducts that could contribute to earlier carcinogenicity in this model. We also prepared hepatic microsomes from male and female p53+/- and wild-type mice exposed to DES or vehicle. Western blot analysis demonstrated modestly higher basal levels of various cytochrome P450 (CYP) enzymes in the untreated p53+/- mice compared to the wild-type mice. Furthermore, P450 levels were higher in female DES-treated p53+/- mice compared to treated wild-type mice. For the p53+/- knockout mice to be used with contidence in drug safety studies, a further understanding of the metabolic capacity of these animals is needed.

Modulation of endometrial transforming growth factor beta (TGFbeta) by tamoxifen.

Transforming growth factor beta (TGFbeta) immunoreactivity was determined in endometria from non-drug-therapy and tamoxifen-treated patients. Sections were scored for pathology and quantity image analysis performed to determine levels of glandular- or fibrosis-associated TGFbeta1. Tamoxifen-treated patients displayed greater levels of endometrial dysplasia and glandular hyperplasia, in addition to a statistically significant (P<0.0001) elevation in gland-associated TGFbeta1 protein.

An appraisal of telomerase activity in benign prostatic hyperplasia.

BACKGROUND: Prostate cancer is one of the commonest neoplasms in elderly males in developed countries. It is not clear which individuals are at high risk of developing aggressive adenocarcinoma of the prostate. Biomarkers are therefore urgently needed to identify such individuals. It had been suggested both by ourselves and others that prostatic telomerase activity may represent a valuable marker in this respect, particularly if applied to BPH, as tissue is readily available from both transurethral resection of prostates and transrectal ultrasound biopsy. METHODS: Tissue was collected prospectively from 46 patients with BPH who underwent TURP for clinically benign prostatic disease, and who were examined using the telomeric repeat amplification protocol (TRAP assay). RESULTS: Telomerase activity was not detected in any of 46 BPH samples, using TRAP assay conditions of 0.12, 1.2, and 12.0 microg protein. CONCLUSIONS: The study confirms that telomerase is not detectable in BPH samples. This would suggest that absence of telomerase activity may be a strong indicator of a lack of cancer. However further studies are necessary to confirm this.

N-demethylation accompanies alpha-hydroxylation in the metabolic activation of tamoxifen in rat liver cells.

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via alpha-hydroxylation leading to modification of the N(2)-position of guanine in DNA and to a lesser extent the N(6)-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N(2)-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or alpha-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, alpha-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the alpha position was confirmed in experiments in which [alpha-D(2)-ethyl]tamoxifen, but not [beta-D(3)-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and alpha-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by (32)P-postlabelling is achieved with >90% labelling efficiency.

Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

DNA adducts in human breast tissue: association with N-acetyltransferase-2 (NAT2) and NAT1 genotypes.

The etiology of human breast cancer is poorly understood, but circumstantial evidence points toward exogenous genotoxins as causative agents; they are believed to exert their carcinogenic action by binding to DNA. Because this binding is often preceded by metabolic activation, it is dependent on the expression and activities of metabolic enzymes of the host. Human mammary tissue samples from 42 women undergoing surgery for breast cancer or reduction mammoplasty were analyzed for DNA adducts by 32P-postlabeling analysis. With the butanol extraction method of DNA adduct enrichment, adduct levels were determined to be 0-414.6 adducts per 10(9) nucleotides, with considerable interindividual variation. To characterize the DNA adducts, we reanalyzed the adduct spots by reversed-phase high-performance liquid chromatography. Of two major adduct spots detected on TLC that accounted for up to 70% of the DNA modification, one eluted as a single peak on high-performance liquid chromatography, whereas the other was resolved into two distinct peaks of radioactivity. These major adducts were highly lipophilic in character. The N-acetyltransferase-1 (NAT1) and NAT2 genes were analyzed for common mutations using random RFLP analysis. An association between NAT2 acetylator status and adduct levels was observed; significantly elevated adduct levels occurred in the mammary DNA from women who were designated slow acetylators for NAT2 [median adduct level = 83.0 adducts per 10(9) nucleotides (range, 9.0-414.6)], as compared with the levels in individuals designated rapid acetylators for NAT2 [median adduct level = 39.7 adducts per 10(9) nucleotides (range, 0-91.0; P = 0.0053)]. On the other hand, NAT1 genotypes were not significantly associated with adduct levels. Although the agents responsible for the DNA modifications in the human breast are not known, this pilot study supports the hypothesis that DNA adduct formation in the human breast may be influenced by the NAT2 genotype.

Comparison of the formation of 8-hydroxy-2'-deoxyguanosine and single- and double-strand breaks in DNA mediated by fenton reactions.

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone. Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions. Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

Lipid peroxidation-induced etheno-DNA adducts in the liver of patients with the genetic metal storage disorders Wilson's disease and primary hemochromatosis.

To assess DNA damage caused by lipid peroxidation due to copper and iron storage disorders in the human liver, the formation of the etheno adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytine (epsilon dC) was measured in liver DNA from normal subjects and from patients with Wilson's disease (WD) and primary hemochromatosis. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in normal liver were 19.3 +/- 4.9 and 27.5 +/- 10.0, respectively. The mean epsilon dA and epsilon dC levels per 10(9) parent nucleotides in WD were 61.03 +/- 7.95 and 91.50 +/- 36.02, and in primary hemochromatosis, they were 46.62 +/- 32.83 and 64.32 +/- 11.55, respectively, two to three times higher than those in the normal liver. The etheno adduct levels were highly correlated with the copper content of the liver in the normal and WD samples. This study demonstrates for the first time the formation of promutagenic etheno adducts in humans in association with copper and iron storage-induced lipid peroxidation. Thus, the etheno adducts are implicated as initiating DNA damage in copper/iron-induced carcinogenesis in humans and should also be explored as biomarkers in disease progression and prevention trials.

Tamoxifen and the female genital tract.

Ugwumadu AHN, Carmichael P, Neven P. Tamoxifen and the female genital tract. Int J Gynecol Cancer 1998; 8: 6-15. Tamoxifen was originally developed by Imperial Chemical Industries (England) (ICI) in 1966 as an anti-estrogenic contraceptive. Ironically, it found a role in the treatment of anovulatory infertility, but its most important application to date is in adjuvant hormonotherapy for breast cancer. Tamoxifen has a complex and poorly understood mix of estrogenic and anti-estrogenic properties with variable and contrasting effects on hormone-sensitive target tissues, such as the endometrium. This article reviews the gynecologic lesions associated with tamoxifen therapy and discusses the merits and acceptability of endometrial surveillance tests and the role of progestogens.