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.

alpha-Hydroxytamoxifen, a metabolite of tamoxifen with exceptionally high DNA-binding activity in rat hepatocytes.

It has been proposed that the antiestrogen tamoxifen induces liver tumors in rats and genotoxic effects in vitro through metabolic activation involving, initially, alpha-hydroxylation of the ethyl group. To test this hypothesis, the extent of DNA adduct formation in primary rat hepatocytes treated with tamoxifen and alpha-hydroxytamoxifen was investigated. Hepatocytes from female Fischer F-344 rats were treated with 1 or 10 microM concentrations of either alpha-hydroxytamoxifen or tamoxifen. DNA was isolated and analyzed for the presence of DNA adducts by 32P postlabeling. Chromatography on polyethyleneimine cellulose thin layer chromatography and reverse-phase high performance liquid chromatography revealed that the same pattern of adducts was formed by both compounds. However, the level of adduct formation was 25 and 49 times greater with alpha-hydroxytamoxifen than with tamoxifen at 1 and 10 microM, respectively. The formation of alpha-hydroxytamoxifen as a metabolite of tamoxifen was demonstrated by mass spectrometric analysis of the extracted culture medium. alpha-Hydroxytamoxifen was found to react with DNA in the absence of metabolizing enzymes. These results demonstrate the involvement of alpha-hydroxylation in the metabolic activation of tamoxifen.

Genotoxicity of human mammary lipid.

We tested the proposition that human mammary lipid contains mutagenic/genotoxic agents that could cause DNA damage in adjacent epithelial cells. Lipid samples from breast tissue surgically removed from 40 women undergoing elective reduction mammoplasty were extracted by a solid-phase procedure. Mutagenicity was observed in Salmonella typhimurium TA98 and TA1538 in 16 of 40 (40%) extracts assayed with rat-liver S9, but not in its absence. No mutagenicity was seen in S. typhimurium TA100 or Escherichia coli WP2uvrA(pKM101). Bacterial mutagenicity correlated with micronucleus-forming activity in a metabolically competent mammalian cell line (MCL-5). This genotoxic activity merits further investigation in relation to the etiology of breast cancer.

Lack of genotoxicity of tamoxifen in human endometrium.

The potential for the anti-breast cancer drug tamoxifen [(Z)-1-[4-[2-( dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] to induce genotoxic damage (DNA adducts) in the human endometrium was investigated in vivo and in vitro. Endometria from hysterectomy patients who were not on tamoxifen were sectioned and maintained in short-term organ culture. The cultures were treated with either solvent vehicle (DMSO), tamoxifen, alpha-hydroxytamoxifen [(E)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3- ol; the major DNA-reactive metabolite in the rat], or benzo(a)pyrene. DNA was isolated and analyzed by 32P postlabeling. Chromatography on polyethyleneimine-cellulose TLC plates revealed DNA adducts in endometria treated with alpha-hydroxytamoxifen identical to those seen previously in the rat liver. However, no adducts were seen from treatment with tamoxifen itself. The viability of the enzyme-metabolizing systems of the endometrial samples was demonstrated by the detection of expected DNA adducts induced by benzo(a)pyrene. Examination by liquid chromatography-mass spectrometry of the explant culture media from endometria treated with tamoxifen revealed the presence of the alpha-hydroxy metabolite in a dose-dependent manner, although apparently at levels insufficient to produce detectable DNA adducts. Endometrial DNA obtained from 18 patients undergoing daily treatment with 10-40 mg tamoxifen for 3 months-9 years was also analyzed. No evidence for any DNA adducts induced by tamoxifen was found in any of the patients examined. These data suggest that the genotoxic events observed with tamoxifen in the rat may not apply to the human endometrium.

Identification of the major tamoxifen-deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen.

The antiestrogenic drug tamoxifen induces liver tumors in rats by a genotoxic mechanism. The key step has been proposed to be the formation of a reactive carbocation from the metabolite alpha-hydroxytamoxifen. This compound reacts with DNA in vitro to a small extent (1 in 10(5) DNA bases), giving products identical to those found in rat liver cells treated with tamoxifen. Now we have prepared the more reactive alpha-acetoxytamoxifen, which reacts with DNA in vitro to a much greater extent (1 in 50 bases). The products of this reaction were subjected to 32P postlabeling and shown by both TLC and reverse-phase liquid chromatography to be identical to those isolated from DNA treated with alpha-hydroxytamoxifen and to those found in the liver DNA of rat hepatocytes treated with tamoxifen or of the livers of rats treated with tamoxifen. The major product was also isolated as the nucleoside and characterized by UV, mass, and proton magnetic resonance spectroscopy. It is an adduct of tamoxifen and deoxyguanosine in which the alpha position of tamoxifen is linked covalently to the exocyclic amino group of deoxyguanosine.

Evidence for the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mouse skin.

Dibenz[a,h]anthracene (DB[a,h]A) and its microsomal metabolites, trans-3,4-dihydro-3,4-dihydroxydibenz[a,h]anthracene (DBA-3,4-diol), trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]anth racene, trans,trans-3,4:10,11-tetrahydro-3,4:10,11-tetrahydroxydibenz[a,h] - anthracene (DBA-3,4,10,11-bis-diol) and trans,trans-3,4:12,13-tetrahydro-3,4:12,13- tetrahydroxydibenz[a,h]anthracene were each applied topically to mouse skin and the epidermal DNA isolated 24 h later. 32P-postlabeling analysis of each of the DNA samples was performed. DNA from mice treated with DB[a,h]A produced an adduct map on TLC consisting of one major and three minor adduct spots. A similar pattern of spots was produced by DBA-3,4-diol. No detectable DNA adducts were produced by trans,trans-3,4:12,13-tetrahydro-3,4:12,13-tetrahydroxy- dibenz[a,h]anthracene, although a single, minor adduct spot was produced by trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]- anthracene. However, DBA-3,4,10,11-bis-diol was found to produce a major single adduct that comigrated on thin layer chromatography with the major adduct produced by both DB[a,h]A and DBA-3,4-diol. In addition, this adduct was present at a level 10 times higher than the corresponding adduct produced by treatment with the parent hydrocarbon. Coelution of the major adducts formed from DB[a,h]A and DBA-3,4-diol with that formed from DBA-3,4,10,11-bis-diol was also demonstrated on reverse-phase high performance liquid chromatography. Thus, we propose that, in mouse skin, the major pathway of DB[a,h]A activation to DNA binding products is via a 3,4-diol to the 3,4,10,11-bis-diol and ultimately to a bis-diol-epoxide (potentially the 3,4,10,11-bis-dihydrodiol-1,2-oxide).

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.

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.

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.

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.

Age-related changes in proteolysis of aberrant crystallin in bovine lens cell-free preparations.

Bovine alpha-crystallin fragments were prepared by H2O2/Cu2(+)-mediated free-radical treatment (which generated 15.1 and 16.6 K Mr fragments) and cyanogen bromide cleavage (which generated 8.4, 13.8 and 16.8 K Mr fragments). Proteolysis of the fragments and native and denatured alpha-crystallin was then compared using bovine lens homogenates prepared from either cortex or cores of lenses obtained from animals of different ages (foetal, 1.5 years and 6.5 years old). In all preparations the fragments were more rapidly degraded than the denatured but uncleaved protein, while the untreated crystallin was only slightly susceptible to proteolytic attack. Both developmental and age-related differences in proteolytic activity towards the aberrant crystallins were detected, most notably a marked age-related decline in the fragment catabolism in preparations obtained from the cores. Should a similar decline occur in human lenses then the changes which we have detected may be important contributary factors towards cataractogenesis.

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.

DNA adducts in human and mouse skin maintained in short-term culture and treated with petrol and diesel engine lubricating oils.

Human and mouse skin samples maintained in short-term organ culture were treated topically with used engine oils from petrol- and diesel-powered vehicles. Mice were also treated topically in vivo for comparison. DNA was isolated and analysed by 32P-postlabelling and the labeled DNA digests were resolved on polyethyleneimine-cellulose tlc sheets. A large number of radioactive adduct spots were observed in DNA from skin treated with the used petrol-engine oil, indicating the formation of adducts by many components of the complex oil mixture. Total adduct levels were similar in mouse skin (both in vivo and in vitro) and in human skin, although qualitative differences in the adduct maps were apparent between the human and mouse skin DNA. Treatment with the used diesel engine oil produced adduct levels no greater than that of control samples in mouse skin (in vivo and in vitro), although significant levels were found in human skin DNA from one donor. The results correlate well with the carcinogenic activity of these oils in experimental animals, helping to substantiate the conclusion that petrol engine oils (but not diesel engine oils) may present a carcinogenic risk to man if appropriate measures to minimise skin contact are not observed.

DNA adduct formation in mice following treatment with used engine oil and identification of some of the major adducts by 32P-postlabelling.

Used engine oil from a petrol-powered vehicle was fractionated by column chromatography into seven parts for which the major polycyclic aromatic hydrocarbon (PAH) components were determined by GC. Topical treatment of mice with the fractions and 32P-postlabelling of the skin DNA resulted in the detection of multiple adduct spots on TLC for some, but not all, of the fractions. The majority of the DNA binding capacity of the used engine oil was possessed by the first three fractions, (equivalent to 25, 15 and 14.5%, respectively) of the adduct forming ability of the unfractionated oil. The chromatographic mobilities of the adduct spots induced by these fractions were compared to those produced by unfractionated used engine oil. In addition, mice were also treated topically with reference PAHs, either singly or as mixtures, dissolved in unused oil at the concentrations at which they were present in the used oil. Comparisons were made between the chromatographic mobilities of the adducts formed in mouse skin DNA by synthetic mixtures with those formed by the used oil. From these data, some of the major adducts produced by treatment with used engine oil are suggested to be formed by reactive metabolites of benzo[b]naphtho[1,2-d]thiophene, benzo[c]phenanthrene, benzo[g,h,i]fluoranthene, chrysene, benzo[a]pyrene and benzo[g,h,i]perylene.

A working in-house computer system for a general ophthalmology practice.

The authors describe their computer system, which was designed to support all of the information needs of their office and utilize the capabilities of the computer in every aspect of the practice. The system is now intricately involved in both clerical and medical activities. Problems of implementation were alleviated by the participation of the physicians and staff in designing the portions of the system with which they would be working; thus, the system is tailored to fit the needs of each individual.

Detection of bulky DNA lesions in the liver of patients with Wilson's disease and primary haemochromatosis.

In the human metal storage disorders of Wilson's disease and primary haemochromatosis, ion transport and excretion dysfunctions result in the intracellular deposition of copper and iron, respectively. These aberrant accumulations of transition metal ions lead to extensive tissue damage, especially in the liver. In order to investigate the possible role of metal ion-mediated oxygen free radical-generated DNA damage in these processes, DNA was isolated from liver of eight Wilson's disease patients and six haemochromatosis patients. Significant levels of bulky DNA damage were detected in these samples by 32P-postlabelling analysis, but were not found in liver DNA from age-matched controls. This form of novel DNA damage was detected in six out of eight Wilson's patients, varying between approximately 1 and 100 base modifications per 10(8) nucleotides, and in all of the haemochromatosis samples examined; the levels of modified species per 10(8) nucleotides varying from approximately 2 to 50. HPLC analysis of these bulky DNA lesions demonstrated that the species formed in Wilson's disease and in haemochromatosis were chromatographically identical but were not the same as putative purine dimers that can be generated in DNA by in vitro incubation with Cu+/Fe2+ and H2O2 (although the possibility that the adducts detected are closely related has not been ruled out). Analysis of the oxidative base lesion 8-hydroxydeoxyguanosine showed that levels were not elevated in liver DNA from either Wilson's disease or haemochromatosis sufferers. In fact, a statistically significantly lower level of this lesion was found in Wilson's disease patients than in controls. These data suggest that bulky DNA damage present in the liver of both wilson's disease and primary haemochromatosis patients may play a more important role in the induction of tissue damage than 8-hydroxydeoxyguanosine. The novel DNA damage detected by 32P-poslabelling may also be a significant factor in the initiation of neoplasia leading to malignant hepatoma in haemochromatosis patients.

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.

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.

Metabolism of crystallin fragments in cell-free extracts of bovine lens: effects of ageing and oxygen free-radicals.

1. The ability of cell-free preparations from bovine lens to degrade fragments of alpha-crystallin has been studied. Crystallin fragments, produced by either chemical cleavage with cyanogen bromide or prolonged treatment with H2O2 and Cu2+ to produce hydroxyl radicals, were labelled with 125I and incubated with preparations obtained from lenses from animals of different age. 2. Results showed that the ability of the preparations obtained from the lens cores (the innermost part of the lens composed of enucleated non-dividing cells incapable of protein synthesis) to degrade crystallin fragments decreased with animal age. No such age-related correlation was obtained with preparations obtained from the cortex (the outer region of the lens surrounding the core). 3. The effect of incubation of the various lenticular preparations with H2O2 and Cu2+ on subsequent ability to catabolise crystallin fragments was also examined. Preparations from the oldest lenses were found to be the least resistant to free-radical attack. 4. The relative susceptibility of the crystallins and non-lenticular proteins to H2O2/Cu(2+)-mediated free-radical attack was examined. Not only were the various crystallins (alpha, beta and gamma) far more resistant to cleavage under these conditions, they also protected the non-lenticular proteins from free-radical-mediated attack. The comparative resistance of the crystallins to attack and their ability to protect other proteins appeared to be dependent on their structural integrity as prior denaturation with acid and/or cleavage with cyanogen bromide eliminated these properties. 5. It is suggested that crystallins (which show sequence homology to some heat-shock proteins) possess homeostatic functions which could protect other proteins (e.g. proteases) from certain forms of free-radical-mediated damage; crystallins may therefore be important in ageing in general where aberrant polypeptides accumulate.