Under which legislation was the information requested (Regulation 793/93 or Directive 67/548/EEC): Regulation 793/93

Transcription

1 1 (16) Conclusion of substance evaluation for transitional dossiers Summary conclusion of evaluation of a substance according to transitional measures described in Article 135(2), 136 (1) or 136(2) and 48 of the REACH Regulation. Substance concerned: 1. Chemical name: 4-tert-butylphenol 2. EC No: CAS No: Member State reviewing the transitional dossier: Norway Under which legislation was the information requested (Regulation 793/93 or Directive 67/548/EEC): Regulation 793/93 The specific decision, reference or regulation number, which relates to this testing request: Commission Regulation (EC) No 506/2007/ and Com. Reg. (EC) No 466/2008 Date of submission by Member State: September 2013 Registration number (if available): 9d8bb7ca-18a5-44ef-e f67d249/DISS-9d8bb7ca-18a5-44ef-e f67d249_DISS-9d8bb7ca-18a5-44ef-e f67d249.html

2 2 CONCLUSION OF TRANSITIONAL SUBSTANCE EVALUATION Need for harmonised classification and labelling Need for identification as SVHC (authorisation) Need for a restrictions proposal Need for other Community-wide measures Such as: substance evaluation process/dossier evaluation Need for action at national level or voluntary action by industry Such as: update of registrations No follow-up action at EU level required Tick relevant box(es) x x

3 3 SHORT JUSTIFICATION FOR CONCLUSION 1) Commission Regulation No 466/2008 required local exposure information on the release from two processing sites (5 and 6) into wastewater treatment plants and aquatic compartment (freshwater and marine) from industry. It is recognised that the exposure information in the EU risk assessment report is no longer relevant since production and use volumes have changed significantly since REACH entered into force. Several companies have submitted registrations dossier for 4-tert-butylphenol in 2010 and ) Commission Regulation No (EC) No 506/2007 requires an endocrine effects study with fish ( draft OECD ext. ELS test). Background In vitro data for ptbp and structurally related chemicals: Para-tert-butylphenol (ptbp) has been identified as a potential endocrine disruptor based on the chemicals ability to interact with and activate the estrogen receptor (ER) in vitro in several organisms and bioassays. These in vitro studies include recombinant cell reporter systems (yeast and bacterial estrogen screens) which express the human estrogen receptor, estrogen dependable MCF7 cancer cells, receptor-binding assays with purified liver homogenates containing ERs and induction of the estrogenic biomarker vitellogenin (VTG) in primary liver cells from fish. Estrogen receptor-binding studies The results from receptor-binding studies show that ptbp, and a range of alkylphenols and other industrial chemicals, are able to bind to the human, rat and fish ERs. The studies with human ERs report that ptbp binds to the ER with times lower affinity than the natural ligand 17β-estradiol (E2) (Olsen et al., 2005). The binding affinity of ptbp to the human ER were 20 times lower than the more hydrophobic alkylphenol 4-tert-octylphenol (ptop) and 500 times

4 4 lower than Bisphenol A (BPA). The binding affinity of ptbp to the human ERs resemble that of the rat ER, where binding affinity of ptbp were times lower than E2, 15 times lower than ptop and 30 times lower than BPA (Blair et al., 2000). Interestingly, ptbp displayed similar binding affinity as the structurally related alkylphenol 4-tert-pentylphenol, ptpp. Studies with rainbow trout liver homogenates demonstrate that ptbp binds to the ER with about times lower binding affinity than E2, and fairly similar binding affinity as ptpp, ptop and BPA (Olsen et al., 2005; Tollefsen and Nilsen, 2008). The available in vitro receptor-binding studies clearly suggest that ptbp binds to the human, rat and fish ER with binding affinities that are comparable to well-characterised endocrine disruptors such as ptpp, ptop and BPA. Activation of the estrogen receptor ptbp has been demonstrated to activate the human and fish ERs. Studies with the yeast estrogen screen, which is stably transfected with the human ER, report that ptbp activate the human ER at approximately 1.4 million times lower potency than E2, 10 times lower potency than ptpp, 1000 times lower potency than ptop and 1500 lower potency than BPA (Routledge and Sumpter, 1997; Sohoni and Sumpter, 1998). Other studies using the estrogen dependent MCF7 cancer cell line, which also express the human ER, confirm that ptbp has a relative low estrogenic potency (0.3-5 million times lower than E2), similar potency as ptpp, about times lower potency than ptop and BPA (Olsen et al., 2005; Soto et al., 1995). Interestingly, induction of the estrogenic biomarker VTG in rainbow trout liver cells (hepatocytes) show that ptbp exhibit a higher relative potency than that reported in bioassays with the human ERs, with a potency times lower than E2, similar potency as ptpp and within one order of magnitude lower potency than ptop and BPA (Olsen et al., 2005; Tollefsen et al., 2008). An interspecies comparison confirms that ptbp display a higher relative ER binding affinity and estrogenic potency in fish than human-derived from in vitro bioassays (Olsen et al., 2005). Structure activity relationship (SAR) studies with a range of alkylphenols with different chain lengths, number of alkylated substituents and substituent position show that ptbp are among the most estrogenic alkylphenols in rainbow trout when tested in vitro

5 5 (Tollefsen et al., 2008). In vivo data and read across to structurally related chemicals No fish in vivo data has to date been provided for ptbp in open literature, so read-across to closely related alkylphenols was conducted. A full life-cycle (2 generation) study with Japanese medaka (Oryzias latipes) exposed to ptpp demonstrated that low µg/l concentrations of ptpp led to abnormal sexual differentiation (LOEC=224 µg/l) and VTG induction (LOEC=51 µg/l) in the F1 generation (Seki et al., 2003). Reports of in vivo endocrine modulating and disrupting effects of ptpp also in common carp (Cyprinus carpio) and fathead minnow (Pimephales promelas) at low µg/l concentrations strengthened the concern that moderately sized parasubstituted alkylphenols such as ptpp and ptbp are likely to cause endocrine disruption in fish (Gimeno et al., 1996; Gimeno et al., 1997; Hagino et al., 2001; Panter et al., 2006; Panter et al., 2002). Studies with other well-characterised estrogenic compounds such as BPA and ptop (see RAR for BPA and ptop) have previously led to proposal for low NOECs for reproductive effects in fish at 16 and 12 µg/l, respectively. ptop is currently identified as an SVHC and included in the candidate list due to endocrine disrupting properties ( see. The requirement for a long term study in fish The in vitro estrogenic properties of ptbp, which were found to be fairly similar to ptpp, ptop and BPA in fish, and read across to in vivo data introduced the requirement for a regulatoryinitiated investigation of potential endocrine disrupting effects in in vivo. A protocol for performing a Partial Life Cycle Test with the Fathead Minnow was therefore developed. The test was conducted as a two tiered approach based on an initial test optimisation and range finding test (pilot study) followed by a full study design (final study). Pilot study: A Partial Life Cycle Test with the Fathead Minnow (Pimephales promelas) A 128-day non GLP pilot study with ptbp to determine test concentrations for the full study and evaluate the suitability of endocrine endpoints to be used in a partial life cycle test with fathead minnow has been performed with ptbp at nominal concentrations of 1, 30, 100 and 500 µg/l. Traditional endpoints of fish early life-stage studies, including effects of the test substance on time to hatch, hatching success, stage specific survival and growth were monitored. Endocrine

6 6 mediated endpoints were evaluated including gonadosomatic index (GSI), sex ratio, observation of secondary sex characteristics, measurement of VTG and histopathology of gonads and gonadal ducts. The protocol was based upon the experimental design presented in ANNEX E, Partial Life Cycle Test (or Extended Early Life-Stage Test) of the OECD Detailed Review Paper on Fish Screening Assays for the Detection of Endocrine Active Substances (OECD, 2004). The study also incorporated procedures in the OECD Guideline for Testing of Chemicals, 210: Fish Early- Life Stage Toxicity Test (OECD, 1992) and ASTM Standard E Standard Guide for Conducting Early Life Stage Toxicity Tests with Fish (ASTM, 1998). The main findings of the pilot study were as follows (nominal concentrations in brackets): 1. Water concentrations: measured water concentrations were (2, 25, 82, 413 µg/l), thus deviated more than 20% from the nominal concentrations (1, 30, 100, 500 µg/l). Suggestions to use the measured, rather than nominal have been proposed and both have consequently been presented for clarity. 2. Hatching success: No effects were observed in the hatching success in any group, but a significant delay in hatching for the 82 µg/l (nominal: 100 µg/l) and 413 µg/l (nominal: 500 µg/l) group was observed according to the Jonkheere-Terpstra trend test (p<0.05). Although the biological significance of these findings was questioned, a need for improved sampling design was proposed to properly assess this effect. 3. Survival of Larvae and Juvenile fish: No significant differences in survival of fish at any fish stage observed. 4. Sex ratio: A significant reduction in male fish and fish displaying male gonads was observed at the 413 µg/l (nominal: 500 µg/l) treatment group according to the Jonkheere- Terpstra trend test (p<0.05). The findings were based on gross internal sex and on external sex determination. 5. Growth: No significant changes in length and weight of females were observed. A

7 7 treatment-related effect on fish length and weight were observed for males at the concentration of 413 µg/l (nominal: 500 µg/l) according to the Jonkheere-Terpstra trend test (p<0.05). No significant differences were identified in males of any treatment group when compared to the control according to the Dunnett s test (p<0.05) 6. Gonadosomatic index (GSI): No significant changes in GSI of either males or females were observed. 7. Vitellogenin (VTG): No significant changes in plasma VTG in males were observed, apparently due to high inter-replicate variations. An apparent treatment-related increase in female plasma concentrations of VTG in the 413 µg/l (nominal: 500 µg/l) group was suggested, although this was not identified to be significant according to either the Jonkheere-Terpstra trend test (p<0.05) nor the Dunnett s test (p<0.05). Large intra and inter-replicate variation was clearly evident in both males and female groups, leading to concern that an apparent trend in reduction of VTG in females at the 2 µg/l (nominal: 1 µg/l) and 25 µg/l (nominal: 30 µg/l) may have been treatment related. No explanation for a non-endocrine mechanism causing this lowconcentration effect could be provided, and thus leading to this observation remained unclear. 8. Onset of male sex characteristics: A treatment related effect on fish displaying at least one male secondary sex characteristic was observed in the 413 µg/l (nominal: 500 µg/l) group. It was not possible to discriminate whether the observed effect were due to a delay in maturation or lack of ability to develop male secondary sexual characteristics, however. 9. Pigmentation on dorsal fin or nose/lip: Pigmentation on the dorsal fin or nose/lip of fish in the treatment groups was not significantly different from the control. However, the fish in the 413 µg/l (nominal: 500µg/l) treatment group displayed an apparent, although non-significant, treatment-related effect. 10. Presence of fatpad and fatpad Score: A treatment-related reduction in the proportion of male fish with a fatpad and in fatpad scores was identified in the 413 µg/l (nominal: 500 µg/l) treatment group according to the Jonkheere-Terpstra trend test (p<0.05). The male fish in the 82

8 8 µg/l (nominal: 100µg/l) treatment also displayed an apparent, although non-significant, treatment-related effect. 11. Presence of Tubercles, Tubercle Count and Tubercle Score: Presence of tubercles (counts and score) of fish in the treatment groups were not significantly different from the control. However, the fish in the 413 µg/l (nominal: 500µg/l) treatment displayed an apparent, although not significant, treatment-related effect. 12. Histopathology: All of the males (12 out of 12) evaluated in the 413 µg/l (nominal: 500 µg/l) treatment group exhibited feminization of gonadal ducts (minimal to moderate). Presence of testicular oocytes (minimal to mild) was observed in 5 out of 12 samples evaluated at the same treatment group. The presence of intravascular fluid in female ovaries, which was considered indicative of increased VTG production, was observed at 82 µg/l (nominal: 100 µg/l) and 413 µg/l (nominal: 500µg/L). The effect occurring at 82 µg/l was not considered statistically significant. Summary assessment of pilot study: It is concluded that the induction of VTG in females, changes in male sex ratio, feminization of male gonads and presence of testicular oocytes (intersex) are clear indicators of endocrine disruption. Observations such as delayed onset of male sex charateristics, pigmentation of fin or nose/lip, reduction in fatpads and/or fatpad scores, and reduction in tubercles, tubercle count and score, and presence of intravascular fluid in female gonads were all considered to provide supportive evidence for an ED mode of action. Delayed hatching, presence of intravascular fluid in female gonads and the presence of fatpads and fadpad score in male fish were identified as being the most sensitive endpoints determined and suggest a NOEC of 25 µg/l (nominal: 30 µg/l) and a LOEC of 82 µg/l (nominal: 100 µg/l). A negative trend in VTG production in females was indicated at even lower concentrations, although an endocrine mechanism to explain the observations could not be provided. It was concluded that the limited test design of the PILOT study could not secure sufficient statistical power to conclude on all of the endpoints tested nor comply fully with test criteria proposed for a full fish test (E ; OECD, 1992, 2004), thus required a full test adopting to a revised and improved test design.

9 9 Full study: A Partial Life Cycle Test with the Fathead Minnow (Pimephales promelas) A GLP study was performed with ptbp at 10, 30, 100 and 300 µg/l for 128 days to determine if exposure of fathead minnows to ptbp during their early development would result in changes typically associated with exposure of fish to estrogenic chemicals. The study was conducted according to the procedure Para-Tertiary Butyl Phenol (PTBP): A Partial Life Cycle Test with the Fathead Minnow (Pimephales promelas) (Krueger et al, 2008). The exposure period included a five-day hatching period followed by up to 123 days of post-hatch development of larvae and juvenile fish. Survival, body weights and lengths, and observations of abnormal behavior as well as endocrine-mediated endpoints including time to hatch, hatching success, observations of secondary sex characteristics, measurement of plasma VTG, measurement of GSI and histopathology of gonads were recorded and results are summarised below. A study summary is available in the disseminated data of the registration dossier: The main findings of the full study were as follows: 1. Water concentrations: The mean measured concentrations in the 10, 30, 100 and 300 µg/l treatment groups were 96, 90, 83 and 85% of nominal concentrations, respectively. Although some single measurements in the 100 and 300 µg/l groups were slightly lower than 80% of nominal, measured concentrations in these groups remained within ±20% of the initial measured concentrations. 2. Hatching Success and Time to Hatch: No effects were observed on hatching success at any of the concentrations tested over the five-day hatching period. A significant delay in the mean time to 50% hatch was observed at the highest concentration (300 µg/l) according to both the Jonkheere-Terpstra trend test (p 0.05) and the Dunnett s test (p 0.05).

10 10 3. Survival of Larvae and Juvenile Fish: A significant reduction in the survival of fish at the 300 µg/l concentration was observed at day 33. The overall high survival rates (>90%) for all groups were proposed to be an indicator of good health and findings proposed to be of low biological relevance. There were no apparent effects on fish survival from day 33 to test termination in any treatment group. 4. Sex Ratio: No significant changes in sex ratio were identified at the concentrations tested. 5. Growth: Treatment-related effects on growth (weight and length) in both males and females were observed in the 30, 100 and 300 µg/l treatment groups according to both the Jonkheere- Terpstra trend test (p 0.05) and the Dunnett s test (p 0.05). No significant change in the condition index of the fish was observed in either males or females. The lack of consistency between the growth parameters (e.g. weight and length) and condition index was proposed caused by a general treatment-related delay in fish development at concentrations higher than 10µg/L. 6. Gonadosomatic Index (GSI): There were no statistically significant differences in the GSI of females or males between the negative control and any of the treatment groups. 7. Vitellogenin (VTG): A treatment-related increase in female plasma VTG concentrations was identified in the 300 µg/l treatment group according to both the Jonkheere-Terpstra trend test (p 0.05) and the Dunnett s test (p 0.05). No statistically significant differences were observed in male plasma VTG concentrations. 8. Male Secondary Sex Characteristics: A treatment-related effect on fish displaying at least one male secondary sex characteristic was observed in the 30, 100 and 300 µg/l treatment groups according to the Jonkheere-Terpstra trend test (p 0.05). Only the 300 µg/l treatment group was significantly different from the control according to the Dunnett s test (p 0.05). 9. Pigmentation on dorsal fin or nose/lip: A treatment-related decrease in the proportion of males with a pigmented spot on the dorsal fin or nose/lip was observed in the

11 11 30, 100 and 300 µg/l treatment groups according to the Jonkheere-Terpstra trend test (p 0.05). Only the 30 µg/l treatment group was significantly different from the control when assessing pigmentation on the dorsal fin, whereas the 300 µg/l treatment group was significant different from control when assessing pigmentation on the nose/lip according to a Dunnett s test (p 0.05). 10. Presence of fatpad and fatpad Score: A treatment-related decrease in the proportion of males with a fatpad was observed in the 30, 100 and 300 µg/l treatment groups according to the Jonkheere-Terpstra trend test (p 0.05). No significant differences was observed between the control and any treatment group according to a Dunnett s test (p 0.05), however. 11. Presence of Tubercles, Tubercle Count and Tubercle Score: A treatment-related decrease in the proportion of males with tubercles, tubercules count and tubercle score were observed in the 30, 100 and 300 µg/l treatment groups. No significant difference was observed between the control and any treatment group according to the Dunnett s test (p 0.05), however. 12. Frequency distribution of male secondary sex characteristics: A treatment-related change in the frequency distribution of the various combinations of secondary sex characteristics was observed in the 30, 100 and 300 µg/l treatment groups according to the Fisher s Exact test. 13. Histopathology: Almost all of the males evaluated in the 300 µg/l treatment group (42 of 45) exhibited feminization of gonadal ducts (minimal to mild) according to a Fisher s Exact test. Presence of testicular oocytes (intersex) in one out of 45 male samples was also recorded. Summary assessment of full study: It is concluded that the induction of VTG in females, complete feminization of male gonads are clear indicators of endocrine disruption. Observations such as delayed onset of male sex characteristics, pigmentation of fin or nose/lip, reduction in fatpads and/or fatpad scores, and reduction in tubercles, tubercle count and score, were all considered to provide supportive evidence for an ED mode of action. It was noted by the contract laboratory that these endpoints showed treatment-related effects that potentially could be related to small delays in development,

12 12 where the overall effect on the fish population level was uncertain. Taking all available information into account, the most sensitive endpoints were reduced growth, reduction in secondary male sex characteristics, and the delay in the time to hatch. Overall statistical LOEC and NOEC values were 30 µg/l and 10 µg/l, respectively. Clearly defined estrogenic effects were clearly present in the 300 µg/l treatment group as evidenced by feminization of gonadal ducts of male fish and elevated levels of plasma VTG in females. Overall conclusion As identification of an endocrine disruptor should be based on a weight-of-evidence (WoE) approach providing causal relationship between an endocrine mode of Action (MoA) and adverse effects (Kortenkamp et al., 2011; Munn and Goumenou, 2013), demonstration of an estrogenic MoA and in vivo effects at the OECD conceptual framework (CF) for ED testing level 4 or 5 (OECD, 2012) is considered essential. A combination of available in vitro and in vivo data from literature on ptbp and compounds with structural or mechanistic (e.g. estrogenic compounds) similarities and data from the pilot and full fish studies have been used to support a WoE approach. The present data clearly demonstrates that ptbp bind and activate human, rat and fish ERs and in this respect display an endocrine MoA. The data from the two fish studies with ptbp shares a resemblance with previously reported studies with ptpp in various fish species by causing adverse effects such as changing male sex ratios (pilot study: 413 µg/l (nominal: 500 µg/l)), feminization of male gonads (Pilot study: 413 µg/l (nominal: 500 µg/l), Full study: 300 µg/l) and causing variable levels of intersex (pilot study: 413 µg/l (nominal: 500 µg/l), Full study: 300 µg/l). A range of endpoints such as delayed onset of male sex characteristics, pigmentation of fin or nose/lip, reduction in fatpads and/or fatpad scores, and reduction in tubercles, tubercle count and score, were considered to provide supportive evidence for an ED MoA in male fish, although the role of other factors such as treatment-related delay of fish development may not be completely disregarded. Clear estrogenic effects consistent with identifying ptbp as an endocrine disruptor was observed at 300 µg/l in the full study, leading to an NOEC for endocrine disruption of 100 µg/l and a LOEC of 300 µg/l. Based on the available fish studies and endpoints of ptbp reported herein, an overall NOEC of 10 µg/l and a LOEC of 30 µg/l on basis of data from the full study is justified.

13 13 It is considered that the current information is sufficient to classify/identify ptbp as an endocrine disruptor, although low-concentration effects observed in the fish studies such as changes in secondary male characteristics and delay in fish development may warrant additional studies to determine if they are mediated by an endocrine MoA. SUMMARY OF INFORMATION REVIEWED Include a brief overview of the new information reviewed in the transitional dossier. Information A pilot study based on Annex E of OECD "Detailed Review Paper on Fish Screening Assays for the Detection of Endocrine Active Substances A partial life cycle test with fathead minnow (Pimephales promelas) Date study was conducted Reason for information request Determine test concentrations and evaluate endocrine endpoints for the full study partial life cycle test Wildlife International, Ltd Concern on endocrine disruption Comments Results from the pilot study will be used in designing a partial life cycle test with fathead minnow (Pimephales promelas) Used to revise PNECaquatic, sediment and soil and update the risk assessment SUPPORTING DOCUMENTS AVAILABLE List documents available to support the conclusion of the substance evaluation e.g. risk assessment addendum or update, Annex XV dossier.

14 14 Title Author Date Comments Risk assessment on p-tert butylphenol Norway 2008 TIMETABLE FOR FOLLOW-UP ACTIONS (IF NECESSARY) Indicate a preliminary timetable, if available, for any follow-up actions proposed. A formal commitment to prepare an Annex XV dossier is made via the Registry of Intentions. Follow-up action Date for completion Who to complete action? Registrants to update their CSR to reflect the conclusion of this evaluation -Norway concludes with an overall NOEC of 10 µg/l and a PNEC of 1 µg/l. Propose ptbp for CoRAP (substance evaluation) June 2014 Registrants References: Blair, R.M., Fang, H., Branham, W.S., Hass, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., Perkins, R., Sheehan, D.M., The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci 54, E , A.S., Standard Guide for Conducting Early Life-stage Toxicity Testswith Fish. American Society for Testing and Materials. European Union Risk Assessment Report, 4,4'-ISOPROPYLIDENEDIPHENOL (BISPHENOL- A); CAS No: ; Complete risk assessment in one document, (February 2010) A European Union Risk Assessment Report, P-TERT-BUTYLPHENOL CAS No: , RISK ASSESSMENT, Final report 2008

15 15 Environment Agency UK: Environmental Risk Evaluation Report: 4-tert-octylphenol Gimeno, S., Gerritsen, A., Bowmer, T., Komen, H., Feminization of male carp. Nature 384, Gimeno, S., Komen, H., Venderbosch, P.W.M., Bowmer, T., Disruption of sexual differentiation in genetic male common carp (Cyprinus carpio) exposed to an alkylphenol during different life stages. Environ. Sci. Technol. 31, Hagino, S., Kagoshima, M., Ashida, S., Effects of ethinylestradiol, diethylstilbestrol, 4-t pentylphenol, 17β-estradiol, methyltestosterone and flutamide on sex reversal in S-rR strain medaka (Oryzias latipes). Environmental Sciences 8, Henry O. Krueger et al 2008: FINAL REPORT PARA-TERTIARY BUTYL PHENOL (PTBP): A Partial Life Cycle Test with the Fathead Minnow (Pimephales promelas) Kortenkamp, A., Martin, O., Faust, M., Evans, R., McKinlay, R., Orton, F., Rosivatz, E., State of the art assessment of endocrine disrupters final report. Munn, S., Goumenou, M., Key scientific issues relevant to the identification and characterisation of endocrine disrupting substances. Report of the Endocrine Disrupters Expert Advisory Group (ED EAG), p. 34. OECD, OECD Guidelines for Testing of Chemicals 210: Fish, Early-life Stage Toxicity Test., OECD Guideline for testing of chemicals. Organization for Economic Cooperation and Development, Paris, France. OECD, Detailed review paper on fish screening assays for the detection of endocrine active substances. Organization for Economic Cooperation and Development, Paris, France. OECD, Draft Guidance Document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Guideline for testing of chemicals. Organization for Economic Cooperation and Development, Paris, France., p. 44. Olsen, C.M., Meussen-Elholm, E.T., Hongslo, J.K., Stenersen, J., Tollefsen, K.E., Estrogenic effects of environmental chemicals: an interspecies comparison. Comp Biochem Physiol C Toxicol Pharmacol 141, Panter, G.H., Hutchinson, T.H., Hurd, K.S., Bamforth, J., Stanley, R.D., Duffell, S., Hargreaves, A., Gimeno, S., Tyler, C.R., Development of chronic tests for endocrine active

16 16 chemicals. Part 1. An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas). Aquat Toxicol 77, Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances. Environ Toxicol Chem 21, Routledge, E.J., Sumpter, J.P., Structural features of alkylphenolic chemicals associated with estrogenic activity. J. Biol. Chem. 272, Seki, M., Yokota, H., Matsubara, H., Maeda, M., Tadokoro, H., Kobayashi, K., Fish full life-cycle testing for the weak estrogen 4-tert-pentylphenol on medaka (Oryzias latipes). Environ. Toxicol. Chem. 22, Sohoni, P., Sumpter, J.P., Several environmental oestrogens are also anti-androgens. J Endocrinol 158, Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Serrano, F.O., The E-SCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environ. Health Perspect. 103, Tollefsen, K.E., Eikvar, S., Finne, E.F., Fogelberg, O., Gregersen, I.K., Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture. Ecotoxicol Environ Saf 71, Tollefsen, K.E., Nilsen, A.J., Binding of alkylphenols and alkylated non-phenolics to rainbow trout (Oncorhynchus mykiss) hepatic estrogen receptors. Ecotoxicol Environ Saf 69,