Meeting report: international workshop on endocrine disruptors: exposure and potential impact on consumers health.

The French Agency for Food, Environmental and Occupational Health and Safety (Anses) hosted a two-day workshop on Endocrine Disruptors: Exposure and Potential Impact on Consumers Health, bringing together participants from international organizations, academia, research institutes and from German, Swedish, Danish and French governmental agencies. The main objective of the workshop was to share knowledge and experiences on endocrine disruptors (ED) exposure and potential impact on consumers' health, to identify current risk assessment practices and knowledge gaps and issue recommendations on research needs and future collaboration. The following topics were reviewed: (1) Definition of ED, (2) endpoints to be considered for Risk assessment (RA) of ED, (3) non-monotonic dose response curves, (4) studies to be considered for RA (regulatory versus academic studies), (5) point of departure and uncertainty factors, (6) exposure assessment, (7) regulatory issues related to ED. The opinions expressed during this workshop reflect day-to-day experiences from scientists, regulators, researchers, and others from many different countries in the fields of risk assessment, and were regarded by the attendees as an important basis for further discussions. Accordingly, the participants underlined the need for more exchange in the future to share experiences and improve the methodology related to risk assessment for endocrine disrupters.

[Assessment of the sensitizing potency of cosmetic ingredients and commodities. How will the ingredients of cosmetics and commodities be tested in Europe today and tomorrow?]. / Bewertung des sensibilisierenden Potenzials von Stoffen in Kosmetika und Bedarfsgegenständen. Wie werden Inhaltsstoffe von Kosmetika und Bedarfsgegenständen heute und morgen in Europa getestet?

Cosmetics and certain commodities are applied or used by consumers directly on the skin. Creams may remain on the skin for longer periods, hair is dyed multiple times per year, nickel ions can be released from studs and piercings in areas of skin damage or migrate from toy materials into the skin of children. Accordingly, using or handling such products always entails a risk for developing a contact allergy. Moreover, daily usage and repeated contacts to certain cosmetics and commodities might lead to repeated elicitation of contact eczema in people already sensitized against allergenic ingredients. Unfortunately, contact allergy is not curable. For the assessment of the allergenic potential of chemicals, only testing based on animal experiments was available in the past. In 2003, the 7(th) amendment of the Cosmetics Directive 76/768/EWG laid down a ban on animal testing of cosmetic ingredients and from 2013 a general marketing ban of such products as well. Therefore, the development and validation of non-animal methods for assessing the toxicological endpoint sensitization/allergenic potency of chemicals is a major task for the years ahead and remains equally a challenge for industry and regulatory agencies.

Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

Study of the sensitising potential of various textile dyes using a biphasic murine local lymph node assay.

Disperse dyes, which are suitable for dyeing synthetic fibres, are responsible for the great majority of allergic contact dermatitis (ACD) cases to textile dyes. The aim of the present study was to investigate the sensitising potential of various disperse dyes using a biphasic protocol of the local lymph node assay (LLNA). Briefly, mice were shaved over a surface of approximately 2 cm(2) on their backs and treated using a "sensitisation-challenge protocol". The shaved surface was treated once daily on days 1-3 with 50 microl of the test solution. Animals remained untreated on days 4-14. On days 15-17, mice were treated with 25 microl of the test solution on the dorsum of both ears. Mice were killed on day 19 with deep CO(2) anaesthesia, the lymph nodes prepared and various end points, such as ear thickness, ear punch weight, lymph node weight, lymph node cell count and the proportion of various lymphocyte subpopulations, were determined by flow cytometry. The results were compared to control group treated with the vehicle alone. Our results showed that almost all of the tested textile dyes caused a significant increase in lymph node cell count and lymph node weight. We also observed an increase in ear thickness and ear punch weight in most of the concentrations tested for various textile dyes. We observed a decrease in CD4+ and CD8+ cells and an increase in CD19+, CD45+ and CD45+/1A+ cells in most of the cases, which is characteristic for allergens. The CD4+/CD69+ cells increased in only few experiments mainly with Disperse Blue 124 and Disperse Blue 106. Based on our results, the disperse dyes could be arranged in four groups on the basis of their sensitising potency in the following decreasing order (in parenthesis: lowest concentration causing a significant increase in lymph node cell number): group 1, strong: Disperse Blue 124 and Disperse Blue 106 (0.003%); group 2, moderate: Disperse Red 1 and Disperse Blue 1 (3%); group 3, weak: Disperse Orange 37 and Disperse Blue 35 (10%); and group 4, very weak: Disperse yellow 3 and Disperse Orange 3 (increase at 30% or no increase at 30%). In conclusion, our study shows that the biphasic LLNA protocol was proficient enough to study the sensitisation potential of tested textile dyes and provides data allowing to discriminate them according to their potency.

[Cosmetic products : Safety aspects]. / Kosmetische Mittel : Sicherheitsaspekte.

The safety of cosmetic products is ensured primarily by the manufacturer. In addition, a set of regulatory elements is effective to exclude the use of hazardous ingredients in cosmetics. In addition to the ban of certain compounds, limitations and use restrictions are compiled in annexes of the directive. In the future, major changes in the safety assessment of cosmetic ingredients are expected considering the ban of animal experiments. The present high level of consumer protection must be maintained; furthermore, innovations should be put into practice without unacceptable risks for the consumer. The new EU regulation on cosmetic products shall be directly applicable in all member states. Finally, two examples for a risk assessment of cosmetic ingredients are presented: diethylene glycol in tooth paste and boric acid in lip care products.

[Cosmetic colorants. Toxicology and regulation]. / Farben in der Kosmetik. Toxikologie und Regulation.

Some recent publications raised concern over a possible link between hair dye use and the incidence of bladder tumours in a Californian population. The Scientific Committee for Cosmetic Products and Non-Food Products intended for Consumers (SCCNFP) demanded the toxicological testing of all hair dyes used in Europe to exclude any risk. The EU commission initiated corresponding measures. Only safe hair dyes will be included on a positive list while all other hair dyes will be banned. The hair dye lawsone--the dyeing ingredient of henna--was evaluated by the SCCNFP as genotoxic but the BfR came to another conclusion. The regulation of both lawsone and henna remains an open question. Furthermore, some cosmetic colorants were critically discussed. The azo dyes CI 12150, CI 26100, CI 27290 and CI 20170 are allowed for use in cosmetics. On cleavage they form the carcinogenic aromatic amines o-anisidine, 4-aminoazobenzene and 2,4-xylidine, respectively. For three of these dyes the cleavage by human skin bacteria in vitro to the respective arylamine was shown experimentally. Further problems may arise from colorants used for tattoos and permanent makeup. These products up to now are not subject to legislation and there are no regulatory stipulations with respect to health safety and purity for colorants used for these purposes.

Harmonisation of rat fetal skeletal terminology and classification. Report of the Third Workshop on the Terminology in Developmental Toxicology. Berlin, 14-16 September 2000.

The initial efforts of the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and the Free University of Berlin to standardise terminology in the field of developmental toxicology began in 1995. Procedures were undertaken to harmonise the terminology used by the International Federation of Teratology Societies (IFTS) and the International Programme on Chemical Safety (IPCS). This article reflects these activities and is a report on the Third Workshop on the Terminology in Developmental Toxicology held in September 2000. This Workshop served as a forum to discuss the results of a survey on the classification of skeletal anomalies that had been previously sent to scientists active in the field. Although high agreement was reached among the evaluators for several terms, the use of a number of terms was rather variable. Therefore, the discussions at the workshop among the experts from research institutions, regulatory agencies, and industry were mainly focussed on those terms for which there was disagreement and/or uncertainties and the possible reasons. Pictures provided by the participants for the illustration of "grey zone" anomalies constituted the basis for detailed discussions. In many of the cases with lower agreement, decisions were facilitated by the provision of the corresponding picture. The main reasons for lower agreement were imprecise terms, insufficient knowledge on postnatal consequences, theoretical terms that are unlikely to occur in isolation, and the possibility of observing a range of severity that might be decisive for the classification of either a malformation or variation. The attendees concluded that "grey-zone" anomalies will never disappear completely and that for the assessment, the grade of severity and/or the frequency of the observation can be decisive for the terminology chosen. A Joint IPCS/IFTS Project was proposed to further consensus of terminology and classification and to link these anomalies to pictures at different skeletal sites. In order to support the harmonisation of regulatory decisions, it was proposed to establish a "Clearinghouse" System under the umbrella of the IPCS. The Clearinghouse could be contacted either by the regulatory authorities or by any company to clarify their queries, particularly with regard to registration or authorisation processes. Finally, it was recommended to also carry out a similar survey on "soft tissue anomalies" and "external findings." The results of this survey will be discussed at a Joint IPCS/IFTS Workshop in Berlin in 2002.

DNA alkylation studies of combined treatment with methylnitrosourea and ethylmethanesulfonate in mice.

The interaction of the DNA-alkylating model compounds, ethylmethanesulfonate (EMS) and methylnitrosourea (MNU), was studied in pregnant NMRI mice by measuring DNA adduction in vivo. Previously, large-scale dose-response studies on teratogenicity as well as on DNA modification were performed using these substances. In addition, the risk of low doses in mice was estimated by comparative use of several approaches including molecular dosimetry. The risk was further analysed by combination experiments on teratogenesis with EMS and MNU. This paper describes a further approach with regard to an interaction of these compounds: the formation of DNA adducts was determined using a combined treatment regimen of [(14)C]-labelled MNU and EMS. The mutual influence of EMS and MNU on the DNA alkylation rates was found to be moderate. The dramatic increase in the teratogenic outcome following combined treatment found in previous studies was obviously not due to a massive interaction regarding the initial DNA alkylation rates. It may be explained, however, by the concept of toxic equivalency. Teratogenesis Carcinog. Mutagen. 20:27-34, 2000.

Formation of a carcinogenic aromatic amine from an azo dye by human skin bacteria in vitro.

Azo dyes represent the major class of dyestuffs. They are metabolised to the corresponding amines by liver enzymes and the intestinal microflora following incorporation by both experimental animals and humans. For safety evaluation of the dermal exposure of consumers to azo dyes from wearing coloured textiles, a possible cleavage of azo dyes by the skin microflora should be considered since, in contrast to many dyes, aromatic amines are easily absorbed by the skin. A method for measuring the ability of human skin flora to reduce azo dyes was established. In a standard experiment, 3x10(11) cells of a culture of Staphylococcus aureus were incubated in synthetic sweat (pH 6.8, final volume 20 mL) at 28 degrees C for 24 h with Direct Blue 14 (C.I. 23850, DB 14). The reaction products were extracted and analysed using HPLC. The reduction product o-tolidine (3,3'-dimethylbenzidine, OT) could indeed be detected showing that the strain used was able to metabolise DB 14 to the corresponding aromatic amine. In addition to OT, two further metabolites of DB 14 were detected. Using mass spectrometry they were identified as 3,3'-dimethyl-4-amino-4'-hydroxybiphenyl and 3, 3'-dimethyl-4-aminobiphenyl. The ability to cleave azo dyes seems to be widely distributed among human skin bacteria, as, under these in vitro conditions, bacteria isolated from healthy human skin and human skin bacteria from strain collections also exhibited azo reductase activity. Further studies are in progress in order to include additional azo dyes and coloured textiles. At the moment, the meaning of the results with regard to consumer health cannot be finally assessed.

Combined prenatal toxicity of 6-mercaptopurine riboside and hydroxyurea in mice.

Hydroxyurea (HU) and 6-mercaptopurine riboside (6-MPr) are used as cytostatic chemotherapeutics. Their teratogenic potential in experimental animals has been well known for several decades. Generally, it is assumed that the toxicity of both agents is due to an interference with enzymes of DNA synthesis. In the case of 6-MPr, it was speculated that the teratogenicity in rodents might be paralleled by or even correlated to an incorporation of 6-thioguanine into the DNA of the embryos. In this study, the interaction between these two compounds with regard to teratogenicity in NMRI mice was investigated. Dose-response data of 6-MPr (s.c.-treatment) were published earlier. First, a dose-response study with HU alone (i.p.-treatment) was performed. From these data, the doses for the combination study were derived: HU 250 mg/kg (NOAEL dose) and 6-MPr 16 mg/kg (strongly teratogenic dose). In all experimental groups the substances were administered to the dams once on day 11 of gestation. Combination effects were investigated applying various dosing regimens. In group I treatment was simultaneous, in group II HU was administered 2 h before 6-MPr, in group III 2 h after 6-MPr. The differences in the overall frequency of gross structural abnormalities were moderate. However, when analysing the effects in more detail (single abnormalities), group III exhibited great differences: 1) 6-MPr co-treatment increased the frequency of HU effects (skull defects) and 2) HU co-treatment decreased the frequency of 6-MPr effects (limb defects) when compared to the findings of the dose-response studies. In addition, the influence of HU on the 6-MPr-induced DNA modification was determined by measuring the incorporation of 6-thioguanine into the DNA of day-11 embryos. As expected, the HU co-treatment corresponding to the group III dosing regimen of the teratogenicity experiment decreased the incorporation rate by ca. 40%. This was in parallel to the decrease in the frequency of 6-MPr effects in the teratogenicity III group. This finding may be considered as a further indication that in the case of 6-MPr, DNA modification is accompanying teratogenicity.

Dose-response relationship of teratogenicity and prenatal-toxic risk estimation of 6-mercaptopurine riboside in mice.

6-Mercaptopurine riboside (6-MPr) is used as a cytostatic chemotherapeutic. The teratogenic potential in rodents has been well known for several decades. In this study, the teratogenic risk of low doses of 6-MPr in NMRI mice was estimated based on a dose-response study. The effective doses corresponding to the incidences of 5%, 1%, and 0.1% were calculated using the probit and Weibull model. The evaluation was performed on the basis of both the fetus and the litter by evaluating the variable all gross structural abnormalities. From these experiments, benchmark doses were obtained which were used in low-dose risk assessment to define a reference dose. Depending on the biometrical model and the statistical unit used, values between 1.9 and 5.2 mg/kg (benchmark ED1) and 3.8 and 6.7 mg/kg (benchmark ED5) were obtained. These values were compared to the no observed adverse effect level (NOAEL) which was determined experimentally. The NOAEL was found to be 5 mg/kg, which is quite similar to the ED5 benchmark doses.

Embryotoxicity induced by alkylating agents: 9. Low dose prenatal-toxic risk estimation of ethylmethanesulfonate based on no-observed-adverse-effect-level risk factor approach, dose-response relationships, and molecular dosimetry.

Analogously to an earlier study using methylnitrosourea (MNU) the prenatal-toxic risk of low doses of ethylmethanesulfonate (EMS) was estimated using different procedures, comparatively. First, the risk of low doses was estimated using linear extrapolation to zero. When using the variable "all gross structural abnormalities" the lowest effective dose in the experiment was 150 mg/kg body wt (5.6% incidence), the additional risk over background was calculated to be 5.0%, and the hypothetical incidence 0.1% was associated with the dose 3 mg/kg EMS. When evaluating "gross structural limb abnormalities," which are not observed in controls, the dose associated with the hypothetical incidence 0.1% was 17.4 mg/kg EMS. Furthermore, derived from a dose-response study of teratogenicity extrapolation to the possible risk of low doses was performed using nonlinear mathematical models. In this case, the results obtained are dependent on the dose response variable as well as from the statistical approach which was chosen. As an example, the values obtained from one evaluation are given: all gross structural abnormalities, Weibull transformation, jackknife approach: ED0.1% = 72 mg/kg EMS. For comparison a "virtually safe dose" was calculated by use of the no-observed-adverse-effect-level (NOAEL) risk factor approach. The NOAEL under our experimental conditions was 100 mg per kg body wt. By using an arbitrarily chosen risk factor of 100 a "safe dose" of 1 mg EMS per kg body wt was obtained. In addition, molecular dosimetry of the DNA adduct rate of O6-ethylguanine in the 11-day-old embryos was used. Based on the assumption that a linear correlation exists between this specific adduct rate and the incidence of teratogenic effects, the hypothetical incidence of 0.1% was associated with a dose of 99 mg/kg EMS. This value is quite similar to that obtained by extrapolation using probit analysis which is in contrast to the results obtained with MNU.

Embryotoxicity induced by alkylating agents: 10. Analysis of the combined teratogenic effects of methylnitrosourea and ethylmethanesulfonate in mice.

In previous studies the direct-acting alkylating model compounds methylnitrosourea (MNU) and ethylmethanesulfonate (EMS) were investigated with regard to dose-response of teratogenicity as well as DNA adduct formation in mice. In this study the teratogenic effects induced by combined treatment with these substances were analyzed using doses which, following single treatment with either substance, were around the threshold level, i.e., no adverse effect level (NOAEL) and lowest observed adverse effect level (LAOEL). Combined treatment of LAOELs resulted in a threshold-like response, while the combination of the NOAEL of one substance with the LAOEL of the other increased the response rate dramatically to nearly 100%. This phenomenon was further evaluated using biometrical methods. The dose-response surface as well as isobolograms were calculated in order to describe the combination effect. In addition, a dose-response model was fitted to the data. In conclusion, the initially surprising high combination effect revealed to be not so extraordinary when considering the steepness of the dose-response relationships of the single substances.

DNA modification induced by 6-mercaptopurine riboside in murine embryos.

The cytostatic drug 6-mercaptopurine riboside (6-MPr) was investigated in mice in order to test the hypothesis that the teratogenicity of this antimetabolite is paralleled by an incorporation into the DNA of the embryos during organogenesis. DNA modification in the embryos was analysed 4 h following s.c. administration of [35S]-labelled 6-MPr to the dams on day 11 of pregnancy. The DNA of the embryos was isolated and hydrolysed to the bases by formic acid. Following separation by cation-exchange HPLC 6-thioguanine was found in the hydrolysate. Quantitation was performed by liquid scintillation counting. Evaluations of 6 doses in the range of 8-25 mg/kg were performed. An incorporation rate of 6-thioguanine from 32-56 pmol per mumol guanine was found in the DNA of the embryos. These findings suggest that, similar to the previously studied alkylating agents, the teratogenicity of 6-MPr may be, at least in part, induced via DNA modification of the embryos.

Embryotoxicity induced by alkylating agents: 8. DNA adduct formation induced by ethylmethanesulfonate in mouse embryos.

In previous studies using methylating agents a correlation was found between the initial DNA adduct rate (O6-methylguanine) in the embryo and the teratogenic efficiency. This was shown by measuring DNA adduct rates in the teratogenic dose range which exhibited similar adduct rates at the equivalent teratogenic dose levels. A similar approach was performed using the ethylating agent ethylmethanesulfonate (EMS). In the teratogenic dose range (150-250 mg/kg bw) the adduct rates of O6-ethylguanine were similar compared to those of O6-methylguanine which were obtained with methylating agents. We conclude that a correlation between teratogenicity and adduct rate (O6-alkylguanine) exists for both methylating and ethylating agents. Furthermore, DNA adduct formation following doses at and below the no-observed-adverse-effect-level (NOAEL) of teratogenicity was determined. The lowest experimental dose was 45 mg/kg EMS. Substantial DNA adduct rates in the embryos were found. These data will be used for molecular dosimetry in a risk assessment of low doses.

Embryotoxicity induced by alkylating agents: 7. Low dose prenatal-toxic risk estimation based on NOAEL risk factor approach, dose-response relationships, and DNA adducts using methylnitrosourea as a model compound.

Prenatal-toxic risk estimation for the alkylating model compound methylnitrosourea (MNU) was performed using different procedures. Risk of low doses was estimated using linear extrapolation to zero (estimated ED0.1%: 0.1 mg/kg body wt MNU) as well as extrapolation by probit analysis based on a dose-response study (estimated ED0.1%: 1.6 mg/kg body wt). Furthermore, a "virtually safe dose" was established by means of the NOAEL risk factor approach (e.g., factor 30:0.03 mg MNU per kg body wt). In previous studies in murine embryos using MNU, we combined dose-response data and DNA adduct rate measurements and deduced that O6-methylguanine is a suitable variable for molecular dosimetry. In a tentative approach, we estimated the teratogenic risk of low doses based on the adduct rates of O6-methylguanine in the DNA of the embryos. It is concluded that in the case of steep dose-response relationships, which are typical for the majority of teratogenic effects, the NOAEL risk factor approach is more conservative than extrapolation based on probit analysis. Risk estimation using dosimetry with this model compound yields estimated incidences similar to linear extrapolation.