Hazard Assessment of Nonylphenol [Nonylphenol, CAS No

Hazard assessment of Nonylphenol [Nonylphenol, CAS No. 25154-52-3 (mixed isomer)]

Theoretically 211 isomers are present in nonylphenol (NP) due to difference in branching of nonyl group and substitution position (Robinson et al., 1976). In this report NP refers to the isomer mixture, unless specifically indicated.

Chemical name : Nonylphenol Synonyms : 4-nonylphenol, NP

Molecular formula : C15H24O Molecular weight : 220.35 Structural formula : OH

C9H19

Appearance : Colorless or Pale yellow liquid1) Melting point : -10°C2) Boiling point : 293 - 297°C1)

20 1) Specific gravity : d 4 = 0.950 Vapor pressure : 0.0032 Pa 1) Partition coefficient : Log Pow = 5.99 (calculated value) 2) Degradability : Hydrolyzability: No Report Biodegradability: Slightly degradable (BOD=0%, 14 days) 3) Solubility : Water, 6.35 mg/l (25°C)2) Soluble in organic solvents, such as benzene, chlorinated solvents, aniline, heptane, aliphatic alcohols, ethylene glycol Amount of Production/ import :12,421t in 1998 (manufactured: 11,644t, imported: 777t)6) Usage : Surfactant (anionic and, non-ion surfactant), stabilizer of ethylcellulose resin, oil-soluble phenol resin, esters, etc.

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NP is contained in processed products such as detergent, oily varnish, rubber solvent and vulcanization accelerator, antioxidant and anticorrosion agent for petrochemical products, antisludge formation for petrochemicals7) Applied law and regulations : Law Concerning Reporting, etc. of Release of Specific Chemical Substances to the Environment and Promotion of the Improvement of Their Management, Water Supply Law, Water Pollution Control Law, Marine Pollution Prevention Law, Sewage Law.

1) HSDB 2001; 2) PHYSPROP, 2000; 3) Tsusansho Koho (Ministry of International Trade and Industry Daily), 1976; 4) Ministry of International Trade and Industry, 1999; 5) The Chemical Daily Co., Ltd, 2000

1. Toxicity Data 1) Information on adverse effects on human health NP demonstrates strong irritation to the eye, skin and respiratory system. If swallowed, weak toxicity is noted (CHRIS, 1984-5). In the case of 2 workers who used a surfactant that contained about 10% of polyoxyethylene octylphenylether and polyoxyethylene nonylphenylether, itchy rash in both hands and forearms was subsequently followed by vitiligo over the skin of both hands, forearms, dorsal pedis, abdomen and hips. As there is a report on decoloration of skin caused by alkylphenols including p-tert-butylphenol and octylphenol, the symptoms is considered to be caused by octylphenol and NP remaining in the surfactant or generated after decomposition (Ikeda et al., 1970).

2) Information on endocrine system and reproductive system (1) in vitro test result related to receptor binding (Attachment-1) Human serum(CDHuS) from which endogenous estrogen was removed by treatment with charcoal-dextran was preserved in 2 types of polystyrene tubes and subsequently

303 Nonylphenol used in a proliferation test of MCF-7 cell (derived from human breast cancer cell, estrogen-sensitive). The activity almost equivalent to 30 pM of 17b-estradiol was observed in CDHuS preserved in one of the polystyrene tubes was used. Furthermore, induction of progesterone receptor was also noted. When the ingredients of 2 polystyrene tubes were extracted with methanol and analyzed, the ingredient characteristically detected in the tube demonstrating the activity, was confirmed as 4-NP (Soto, et al., 1991). Cell proliferating activity is observed in an experiment in which MCF-7 cells fortified with 10-10 - 10-5M of 4-NP was incubated for 5 days. Similarly, weak proliferating activity was noted when 4-NP at 10-6M had been added to ZR-75 cells derived from human breast cancer cell and incubated for 8 days (White, et al., 1994). It was demonstrated that straight chain NP and NP mixture have a weak binding affinity to some receptor protein in rat uterine cytoplasm fractionation or recombinant human estrogen receptor (Binding affinity of these chemicals are 1/680-1/71,000 of that of 17b-estradiol(E2)) (Blair et al., 2000; CERI, 2001). In yeast two-hybrid assay, activation of reporter gene was noted in the branched chain NP (1/670 of that of E2) and no activity was observed in the straight chain NP (Nishihara, 2000). Based on a reporter gene assay using animal cells, both NP mixture (straight chain and branched chain) and straight chain 4-NP induced activation of a reporter gene mediated by estrogen receptor, but the activity of the former was reported to be higher than that of the latter (Balaguer et al., 1999). In a reporter gene assay in which reporter gene responsive to estrogen, plasmid pEREBLCAT gene, luciferase gene and murine estrogen receptor gene were incorporated into MCF-7 cells, 4-NP at 10-6 - 10-5 M induced the transcription activity of a reporter gene mediated by mouse estrogen receptor gene (Balaguer et al., 1999; Legler et al., 1999; White et al., 1994) Legler et al. reported the transcription activity of reporter gene assay by NP is 1/43,000 of that of E2 . Furthermore, NP mixture also induced the transcription activity of a reporter gene mediated by estrogen receptor gene in a reporter gene assay using HeLa cells transfected with rat or human estrogen receptor gene (1/16,000 or less of that of E2)(Yamasaki et al., 2001; CERI, 2001). NP was reported to demonstrate the comparable potential as that of genistin in the

304 Nonylphenol expression of pS2 and TGFb3 in an experiment utilizing expression of endogenous estrogen responsive genes (pS2, TGFb3, monoamine oxidase A, a1-antichemotripsin) (Jorgensen, 2000).

(2) in vivo test results in mammals (Attachment-2) NP has been investigated by many uterotrophic assays, as a screening test method to detect estrogen or ant-estrogen activity (based on the draft OECD guideline). After oral and subcutaneous administration of 4-NP (³ 85%, branched mixture) at 0, 25, 50, 100 and 200 mg/kg/day to female Long Evans rats (21 days old) for 3 days, increase in uterus weight was observed at ³ 50 mg/kg/day and ³ 100 mg/kg/day, respectively (Laws et al., 2000). After oral administration of 4-NP (³ 85%, branched mixture) to ovariectomized female Long Evans rats (60 days old) at 0, 25, 50 and 100 mg/kg/day for 3 days, weight of uterus increased at 100 mg/kg/day (Laws et al., 2000). After oral administration of 4-NP (branched mixture) to female AlpK rats (22 - 23 days old) at 0, 37.5, 75, 150 and 225 mg/kg/day for 3 days, increase in uterus weight was observed at ³ 75 mg/kg/day. When NP at 250 mg/kg/day was administered to AlpK rats and SD rats (22 - 23 days old) for 3 days, increase in uterus weight was observed in both strains of rats as compared to the control. When oral administration of 4-NP (branched mixture) at 0 and 100 mg/kg/day was conducted for 11 days in female AlpK rats (6 - 7 weeks old) which had been ovariectomized at 4 - 5 weeks old, increase in uterus weight was observed in the 100 mg/kg/day group, but no increase was noted in the AlpK female rats (6 - 7 weeks old) subcutaneously treated for 11days with 4-NP (branched mixture) at 0, 0.037 and 27.2 mg/kg/day (continued administration into implanted miniature pump). A slight increase of mammary gland lobule and cell count occurred in the 100 mg/kg/day group after oral administration of 4-NP (branched mixture) at 0 and 100 mg/kg/day to female AlpK rats (5 - 6 weeks old) for 11 days. There was no difference in rat uterus weight between the strains, between the administration periods and between the administration routes. It was reported that the effect on mammary gland was milder in comparison with that observed in the uterus (Odum et al., 1999). When the extract of polystyrene tube (the presence of 4-NP mixture was confirmed) at 1 - 50 mg/rat was subcutaneously administered to female SD rats twice after 19 days

305 Nonylphenol and 20 days of ovariectomy, significant increase in the mitotic index of endometrium was observed at ³ 20 mg/rat (Soto et al., 1991). After subcutaneous administration of 4-NP at 0, 2, 20 and 200 gm/kg/day for 3 days to female SD rats (20 days old), increase in uterus weight was observed in the 200 mg/kg/day group (Yamasaki et al., 2001). After subcutaneous administration of 4-NP to newborn female SD rats from day 1 to 5 of birth, abnormal estrus cycle, decreased fertility, and increased atretic follicles and decreased yellow bodies in the ovary were observed at 500 mg/kg/day (Nagao et al, 2000). After single intraperitoneal administration of NP at 0, 1, 2 and 4 mg/rat to female SD rats (20 - 21 days old), uterus weight, protein content, DNA content and peroxidase activity all increased depending on the dose, and these activities were reported to be 1/1,000 - 1/2000 of that of 17b-estradiol. On the other hand, no effect was noted in the female SD rates (20 - 21 days old) after intraperitoneal administration of NP at 0, 1, 2 and 4 mg/rat simultaneously with ICI 182,780 that is an estrogen receptor antagonist (Lee & Lee, 1996). As mentioned above, there is evidence that NP has estrogenic activity after oral administration at 50 mg/kg or more by uterotrophic assay to assess estrogenic or anti- estrogenic activity.

Many reports have also been available indicating effects of NP on the reproductive system of male rat. After oral administration of 4-NP at 0, 100, 250 and 400 mg/kg/day to male SD rats (12 weeks old) for 10 weeks, atrophy of seminiferous tubule at ³ 100 mg/kg/day, decreased epididymis weight at ³ 250 mg/kg/day, and decreased testis weight and decreased number of sperms at 400 mg/kg/day were noted (De Jager et al., 1999a; 1999b). After subcutaneous administration of 4-NP at 0 and 500 mg/kg/day to newborn male SD rats from day 1 to 5 of birth, germ cells in the seminiferous tubule decreased at 500 mg/kg/day, but no effect was observed on the mobility of sperm and plasma testosterone concentration (Nagao et al., 2000). After intraperitoneal administration of 4-NP at 0 and 8 mg/kg/day to newborn male AlpK rats from day 1 to 10 of birth, no effect was noted on the weight of epididymis, prostatic ventral lobe and testis (Odum et al., 2000).

306 Nonylphenol

When intraperitoneal administration of 4-NP at 0, 0.08, 0.8 and 8.0 mg/kg/day was conducted in newborn male SD rats from day 1 to 15 of birth, and autopsy was conducted when the rats were 31 days old, the weights of testis, epididymis, seminal vesicle and prostatic ventral lobe decreased at ³ 0.8 mg/kg/day, and anus-genital projection distance (AGD) was reduced at 8.0 mg/kg/day. When NP at 8.0 mg/kg/day was administered to SD rats for 18 days when they were aged 1 day, 6 days and 13 days, and the rats were autopsied when they were 31 days old, the weights of testis, epididymis, seminal vesicle and prostatic ventral lobe decreased in the 1-18 day group and 6-24 day group, but no effect was noted in the 13-30 day group (Lee, 1998). When intraperitoneal administration of NP at 8.0 mg/kg/day alone as well as NP at 8.0 mg/kg/day concomitantly with ICI 182,780, an estrogen receptor antagonist, was conducted in newborn male SD rats from day 1 to 5 of birth, the decreased weights of testis, epididymis, seminal vesicle and prostatic ventral lobe observed by administration of NP alone was not observed in the presence of ICI 182,780, indicating that the effect attributable to NP is mediated by estrogen receptor (Lee, 1998). After intraperitoneal administration of NP at 8.0 mg/kg/day in newborn male SD rats from day 1 to 10 of birth, undescended testis accompanied with atrophy was noted (54 - 62%). The incidence was higher on the left testis (22 - 41%), but bilateral incidence was low (11 - 15%). However, concomitant administration of ICI 182,780 nullified such effect (Lee, 1998). During pregnancy (from gestational day 7) and lactation (21 days alter firth) parent

SD rats were given diets containing 0, 25, 500, or 2000 ppm NP. After lactation F1 was administered with the same diet until postnatal day 77. Although dams of groups in 25 ppm or more consumed 9 to 25% less food, gestation period, birth weight, sex ratio of live pups, and number of live or dead pups per litter did not differ between treatment groups. Body weight of offspring in 25 ppm or more (male) and in 2000 ppm (female) and food consumption in the 2000 ppm (male) were decreased as well as increase in intake of water and saline water in 2000 ppm group (male and female) (Ferguson et al., 2000). When NP at 0, 200, 650 and 2,000 ppm (corresponding to 9 - 35, 30 - 100 and 100 - 350 mg/kg/day, respectively) in diet was given to male and female SD rats in a 4- generation reproduction test, no effect was observed in the 200 ppm group, but effects suggesting estrogenic activity such as increased uterus weight, premature vaginal opening,

307 Nonylphenol decreased ovary weight, decreased sperm concentration, were observed at ³ 650 ppm (NTP, 1997; Chapin et al., 1999). After oral gavage of NP at 0, 2, 10 and 50 mg/kg/day to male SD rats for 12 weeks and to female SD rats from 2 weeks before mating throughout gestation, delivery and lactation period to female SD rats, increased weights of liver, kidney and pituitary, decreased thymus weight and increased serum TSH (thyroid stimulating hormone) concentration in F0 males of the 50 mg/kg/day group, decreased ovary weight in F0 females of 50 mg/kg/day group and decreased F1 survival rate (day 0 and 4 of birth) in the males and females of 50 mg/kg/day group were observed. Increased weights of liver and kindney, increased serum FSH (follicle stimulating hormone) concentration and decreased

T3 (triiodothyronine) concentration (day 22 of birth) were observed in the F1 males of 50 mg/kg/day group, and decreased ovary weight, premature vaginal opening, decreased LH (luteinizing hormone) and TSH concentration, and increased T3 concentration (day 22 of birth) were observed in the F1 females of 50 mg/kg/day group, while the number of implantations and surviving offspring (F2) decreased both in the males and females of 50 mg/kg/day group (Nagao et al., 2001). In the final report of the subpanel of NTP on the low dose effects (October 2000) that investigated environmental estrogens and estradiol discussed the low dose effects of NP (released on May 14, 2001) on (NTP, 2001). According to the unpublished multi- generation reproduction test (details are unknown because the type of study, animals used and name of researcher are not stated in the final report of panel), some effect was observed in the F1 generation after 25 ppm was mixed in the diet and given to the animals. To be more precise, increased proliferative activity of T lymphocyte in the spleen stimulated with anti-CD3 antibody, increased relative weight of thymus, decreased volume of SDN-POA in the preoptic area of hypothalamus in the males and delayed estrus in the females were reported. Though the report suggested possibility of effect of NP on immune system and central nerve system, there is no report that indicates similar phenomenon at a dose lower than the above-mentioned level. Compared with the dose at which some effect was noted in animal experiments, the exposure level in humans is much lower, in the order of ng/kg/day. In this regard, the subpanel concludes that the low dose effects of NP is questionable.

308 Nonylphenol

3) Information on general toxicity (1) Acute toxicity (Table 1) Acute toxicity of NP to experimental animals by oral administration is medium and the LD50 is 1,231 mg/kg for mice, 1,300-2,462 mg/kg for rats and not less than 2,000 mg/kg for rabbits (Gaworski et al., 1979; Berol Kemi, 1982; Smyth et al., 1962; 1969; Monsanto, 1978; De Jager et al., 2001). As symptoms for the poisoning, sedation, fouling of hair, motor ataxia and nosebleed have been observed (Berol Kemi, 1982).

Table 1 Acute toxicity test results Mouse Rat Rabbit

Oral LD50 1,231 mg/kg* 1,300 – 2,462 mg/kg* －

Inhalation LC50 －－－

Percutaneous LD50 －－ >2,000 mg/kg *: There are differences between the literature cited.

(2) Repeated-dose toxicity (Attachment-3) When oral administration of NP at 0, 4, 15, 60 and 250 mg/kg/day was conducted for 28 days in male and female SD rats (6 weeks old), the relative weight of liver increased in the males of 60 mg/kg group, while salivation, decrease of body weight increase, increased urine volume, decreased specific density of urine, increased relative and absolute weights of liver, dilation of cecum, centrilobular hypertrophy of hepatocyte, basophilic changes in the proximal renal tubule, basophilic changes and dilation of collecting duct in the kidney and hyperplasia of transitional epithelium of bladder were observed in the males and females of 250 mg/kg. Also in the males of 250 mg/kg group increased urea nitrogen and inorganic phosphorus, decreased chlorine, and increased relative and absolute weights of kidney were noted. As to the females in the 250 mg/kg group, scattering white dots and swelling in the kidney, necrosis of epithelial cells of proximal renal tubule, inflammatory cell infiltration in the interstitium, urinary cylinder, hyperplasia of renal pelvis mucosa as well as dilation of renal pelvis were observed. However, all these changes were reversible. The no-observed effect level (NOEL) in the males and in the females are assessed to be 15 and 60 mg/kg/day, respectively (Ministry of Health & Welfare, 1996). When NP at 0, 200, 650 and 2,000 ppm (corresponding to 0, 15, 50 and

309 Nonylphenol

150 mg/kg/day respectively) mixed in diet was administered to male and female SD rats for 90 days, weight of kidney increased (but returned to the normal level after 4 weeks of recovery test upon completion of administration) and hyaline droplets in the tubular epithelium of kidney decreased in the males of 2,000 ppm group. However, nothing abnormal was noted in the weights of endocrine organs, estrus cycle test and sperm test. The authors estimated the no-observed adverse effect level (NOAEL) as 650 ppm (corresponding to 50 mg/kg/day) (Cunny et al., 1997).

4) Information on mutagenicity/genotoxicity and carcinogenicity (1) Mutagenicity/genotoxicity (Table 2) As to the in vitro studies, the results were negative regardless of the presence or absence of metabolic activation in a reverse mutation test using Salmonella typhimurium, Eschericia coli and yeast, as well as in a chromosome aberration test using CHL cells (Ministry of Health & Welfare, 1996; Shimizu et al., 1985, German Chemical Society, 1988). There is no report on an in vivo study.

Table 2 Results of mutagenicity/genetic toxicity tests Test method Test conditions Result* References in vitro Reverse mutation Salmonella typhimurium TA98, TA100, TA1535, German test TA1537, TA1538 S9(+/-) 5,000 µg/plate － Chemical Society, 1988 Salmonella typhimurium TA98, TA100, TA1535, MHW, 1996 － TA1537 and E. coli WP2uvrA S9(+/-) 200 µg/plate Salmonella typhimurium TA98, TA100, TA1535, Shimizu, et － TA1537 and E. coli WP2uvrA S9(+/-) 100 µg/plate al., 1985 Chromosome CHL cell, S9(+/-) 60 µg/ml － MHW, 1996 aberration test * -: Negative

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(2) Carcinogenicity (Table 3) No carcinogenicity test of this substance was conducted. Table 3 Carcinogenicity assessment by national and international organizations Organs Classification Significance References EPA － No evaluation IRIS, 2002 EU － No evaluation ECB, 2000 NTP － No evaluation NTP, 2000 IARC － No evaluation JARC, 2001 ACGIH － No evaluation ACGIH, 2001 Japan Society for － No evaluation Japan Society for Occupational Health Occupational Health, 2001

5) Information on immune system No report has been made on the effect of NP on the immune system.

6) Fate and Metabolism When the percutaneous penetration and absorption of NP labeled with 14C (the labeling position is unknown) were investigated using the skin of human, pig and rat, the percutaneous penetration and absorption after 8 hours from administration was <5% and <1% respectively regardless of animal species. NP was mainly present in the corneal layer of skin tissue (Monteiro-Riviere, 2000). When NP whose benzene ring was labeled with 14C was orally or intraperitoneally administered to rats, 19% and 70% of the radioactivity were detected in the urine and feces respectively in each case, but no radioactivity was detected in carbon dioxide in the exhaled air. Major urinary metabolite is glucronic acid conjugate (Figure 1) (German Chemical Society, 1988). The result of an in vitro experiment indicated that NP is subject to sulfate conjugation by human sulfate transferase. Sulfate conjugation was also noted in an experiment in which NP and sulfuric acid were added to HepG2 cells derived from human liver cancer, suggesting sulfuration of NP in the living body (Suiko et al., 2000).

グglucronicルクロ acidン酸 conjugate抱合体 C9H19 OH 硫sulfate酸抱合conjugate体

ノニNonylphenolルフェノール Fig. 1: Metabolic pathway of nonylphenol

311 Nonylphenol

2. Hazard assessment at present There is no report on the effect of NP on human endocrine system and reproductive system. In the in vitro experiments conducted to investigate the effect of NP on endocrine system, NP demonstrated weak estrogenic activity (Receptor binding affinity is 1/680 – 1/71,000 of that of E2 and transcription activation capability 1/670 or less of that of E2.) and the affinity of branched NP tended to be higher than that of normal NP. NP showed weak estrogenic activity at in vivo uterotrophic assay using immature or ovariectomized rats (The effect was observed by oral administration at the doses of 50 mg/kg/day or more.). Furthermore, the effects mediated by estrogen receptor were also observed in a test in which intraperitoneal administration of NP and an estrogen receptor antagonist was conducted in newborn male SD rats. As the effects on reproductive system, decreased number of sperms in the epididymis of F2 and premature vaginal opening in F1 - F3 were observed at > 650 ppm (corresponding to 30 - 100 mg/kg/day) in a 4-generation reproduction test using SD rats.

In a 2-generation reproduction test using SD rats, decreased ovary weight of F0 females, decreased survival rate of F1 males and females, decreased ovary weight of F0 female, premature vaginal opening and decreased number of implantations in F1 females as well as decreased number of surviving offspring (F2) were observed at 50 mg/kg/day, indicating the reproductive and developmental toxicity of NP at around 50 mg/kg/day or above. As the information related to the harmful properties, NP is reported to strongly irritate human eye, skin and respiration system. Its effect on the liver and kidney was noted in a repeated-dose toxicity test in rats. Though the results of in vitro mutagenicity tests were negative, no in vivo test results have been reported. There is no report on the carcinogenicity of NP in humans. Nor any carcinogenicity test using experimental animals has been conducted. Most of the reports cited in this assessment document were based on the mixture whose main ingredient was 4-NP. Due to lack of information on these isomers, it is not possible to clarify the effects of each isomer.

3. Risk assessment and other necessary future measures NP has weak estrogenic activity (Its receptor binding affinity is 1/680－1/7,100 of

312 Nonylphenol that of E2 and transcription activation capability is 1/670 or less of that of E2 and it is positive in the uterotrophic asay at the doses 50/mg/kg/day or more.). Reproductive & developmental toxicity was observed at the doses of approximately 50 mg/kg/day or more in rats. As sufficient scientific findings from the in vitro and in vivo test data were already obtained to assess the effect of NP as mixture on the endocrine system and reproductive system, there seems to be no need to plan additional tests in the future. Since adverse effects have been observed for NP due to reproductive & developmental toxicity from existing findings, risk assessment based on hazard and exposure assessment is to be conducted, and appropriate risk management is to be considered.

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Base. Report ISS AMRL-TR-79-11 NTIS AD-A067-31-3. German Chemical Society (1988) Nonylphenol, BUA Report, No.13. IARC (2001) IARC Monograph on the Evaluation of Carcinogenic Risks to Humans. http://www.iarc.fr Ikeda, M., Ohtsuji, H. and Miyahara S. (1970) Two cases of leucoderma, presumably due to nonyl- or octylphenol in synthetic detergents. Ind. Health 8, 192 - 196. IPCS (1995) International Chemical Safety Cards. Jorgensen, M. (2000) Assaying estrogenicity by quantitating the expression levels of endogenous estrogen-regulated genes, Environ. Health Persp., 108, 403. Laws, S.C., Carey, S.A., Ferrell, J.M., Bodman, G.J. and Cooper, R.L. (2000) Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxcol. Sci., 54, 156 -167. Lee, P. C. (1998) Disruption of male reproductive tract development by administration of the xenoestrogen, nonylphenol, to male newborn rats. Endocrine, 9, 105-111. Lee, P.C. and Lee, W. (1996) In vivo estrogenic action of nonylphenol in immature female rats. Bull. Environ. Contam. Toxicol., 57, 341-348. Legler, J., van den Brink, C.E., Brouwer, A., Murk, A.J., van der Saag, P.T., Vethaak, A.D. and van der Burg, B. (1999) Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol. Sci. 48, 55 - 66. Monsanto (1985) Monsanto Industrial Chemicals Co. FYI-OTS-0685-0402 FLWP. Seq. G. Material Safety Data Sheet. Washington, DC: Office of Toxic Substances, U.S. Environmental Protection Agency. Monteiro-Riviere, N.A. (2000) Comparative in vitro percutaneous absorption of nonylphenol and nonylphenol ethoxylates (NPE-4 and NPE-9) through human, porcine and rat skin , Toxicol. Industr. Health, 16, 49 - 57. Nagao, T., Saito, Y., Usumi, K., Nakagomi, M., Yoshimura, S. and Ono, H. (2000) Disruption of the reproductive system and reproductive performance by administration of nonylphenol to newborn rats. Human Exp. Toxicol., 19, 284 - 296. Nagao, T., Wada, K., Marumo, H., Yoshimura, S. and Ono, H (2001) Reproductive effects of nonylphenol in rats after gavage administration: a two-generation study. Reprod. Toxicol., 15, 293-315. Nishihara, T., Nishikawa, L., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M., Takatori, S., Kitagawa, Y., Hori, S. and Utsumi, H. (2000) Estrogenic activities of 517 chemicals by yeast two-hybrid assay. J. Health Sci., 46, 282 - 298. NTP(1997) Final Report on the reproductive toxicity of nonylphenol (CAS #84852-15-3)

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administered by gavage to Sprague-Dawley rats. R.O.W.Sciences 8989-30. NTP (2001) Final Report of the Endocrine Disruptors Low-Dose Peer Review, published in May 14th, 2001. NTP (2000) U.S. Department of Health and Human Services Public Health Service, National Toxicology Program, 9th Repoprt on Carcinogens. Odum, J, Pyrah, I.T., Soames, A.R., Foster, J.R., Van Miller, J.P., Joiner, R.L. and Ashby, J. (1999) Effects of p-nonylphenol (NP) and diethylstilboestrol (DES) on the Alderley Park (Alpk) rat: Comparision of mammary gland and uterus sensitivity following oral gavage or implanted mini-pumps. J. Appl. Toxicol., 19, 367 - 378. Odum, J. and Ashby, J. (2000) Neonatal exposure of male rats to nonylphenol has no effect on the reproductive tract. Toxicol. Sci., 56, 400 - 404. PHYSPROP (2000) Syracuse Research Corporation Physical Properties Database, (http://esc.syrres.com/interkow/PhysProp.htm). Robinson, R.W., Harary, F., and Balaban, A.T. (1976)The numbers of chiral and achiral alkanes and monosubstituted alkanes. Tetrohedron. 32, 355-361. Shimizu, H., Suzuki, Y., Takemura, N., Goto, S. and Matsushita, H. (1985) The results of microbial mutation test for forty-three industrial chemicals. J. Ind. Health, 27, 400-419. Smyth, H.F. Jr., Carpenter, C.P., Weil, C.S., Pozzani, U.C., Striegel, J.A. (1962) Range-finding toxicity data: list VI. Am Ind Hyg Assoc J 23, 95-107. Smyth, H.F. Jr., Carpenter, C.P., Weil, C.S., Pozzani, U.C., Striegel, J.A., Nycum, J.S. (1969) Range-finding toxicity data: List VII. Am Ind Hyg Assoc J 30, 470-6. Soto, A.M., Justicia, H., Wray, J.W. and Sonnenschein, C (1991) p-Nonyl-Phenol: an estrogenic xenobiotic released from “modified” polystyrene. Environ. Health Perspect., 92, 167- 173. Suiko, M., Sakakibara, Y. and Liu, M. (2000) Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases, Biochem Biophys. Res Commun., 267, 80 - 84. White, R., Jobling, S., Hoare, S.A., Sumpter, J.P. and Parker, M.J (1994) Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology, 135, 175-182. Yamasaki, K., Takeyoshi, M., Yakabe, Y. , Sawaki, M., Imatanaka, M. and Takatsuki, M. (2002) Comparison of reporter gene assay and immature rat uterotrophic assay of twenty-three chemicals. Toxicology, 170, 21-30. The Chemical Daily Co., Ltd. (2000) 13901 Chemical Products. CERI (Chemicals Evaluation and Research Institute, Japan) (2001): Report on evaluation and method development for hormone-like effects of exogenous substances. 2000 Contract investigation/research on environment-compatible technology development on behalf of the Ministry of Environment and Industry.

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Ministry of Health, labour and Welfare, Japan (1996) Toxicity Testing Reports of Environmental Chemicals, Chemicals Investigation Promoting Council, Supervised by Office of Chemical Saftey Evaluation and Licensing Div., Pharmaceutical and Medical Safety Bureau, 4, 749 - 772. Tsusanshou Koho (Daily) 1976. Ministry of International Trade and Industry (1999): Survey on the production/import of existing chemical substances in 1998 Japan Society for Occupational Health (2001): Advice on the tolerance limit. San Ei Shi, 43: 95- 119.

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Attachment-1 Results of in vitro test on receptor binding Item Test methods and conditions Result Remark References Binding test Method: Competitive binding test IC50: The binding affinity Blair et al., to ER using [3H]-E2 as a ligand, Straight chain NP: is weak. 2000 Receptor: ER derived from rat 2.8 ´ 10-5 M Straight chain NP: uterine cytoplasm NP mixture: (1/31,000 of that of 2.4 - 4.73 ´ 10-6 M E2) (E2: 8.99 ´ 10-10 M) NP mixture: (1/5,300 – 1/2,700 of that of E2)

Binding to human ER (recombinant NP mixture: The binding affinity CERI, 2001 ER a ligand domain) IC50: 9.5 ´ 10-7 M is positive. (E2: 1.4 ´ 10-9 M) NP mixture: RBA: 0.14％ (1/680 of that of 4-NP Straight chain: E2) IC50: >10-4 M Straight chain 4- (E2: 1.4 ´ 10-9 M) NP: 4-NP mixture: (1/71,000 of that of IC50: 1.3 ´ 10-6 M E2) 4-NP mixture: (E2: 1.4 ´ 10-9 M) (1/930 of that of RBA: 0.11％ E2) The binding affinity is weak. Yeast two Cells: Yeast cell transfected with REC10: No effect of gene Nishihara et hybrid assay Gal4 DNA binding domain/human NP Straight chain: transcription al., 2000 ER ligand binding domain gene, >10-３ M activity in straight Gal4 activation domain/coactivator NP Branched chain: chain NP; Positive TIF2 gene and b-galactosidase 2 ´ 10-7 M in gene reporter gene (E2: 3 ´ 10-10M) transcription activity in branched chain NP(1/670 of that of E2) Reporter Cells: MCF-7 cells transfected with The expression of Positive in gene White et al., gene assay reporter plasmid pEREBLCAT estrogen receptor gene transcription 1994 using gene, luciferase gene and murine was induced at 10-6 - 10-5. activity recombinant estrogen receptor gene cultured cells Reporter gene assay using HeLa Both NP mixture and Positive in gene Balaguer et cells and MCF-7 cells transfected 4-n-NP demonstrated transcription al., 1999 with estrogen receptor gene activity. activity Reporter gene assay mediated by EC50 : 2.6 ´ 10-7 M Positive in gene Legler et al., estrogen receptor (E2: 6 ´ 10-12 M) transcription 1999 Cells: T47D cells transfected with activity (1/43,000 estrogen response sequence and of that of E2) luciferase gene

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Item Test methods and conditions Result Remark References Cells: HeLa cells transfected with PC50: Positive in gene CERI, 2001 ER response sequence and human NP mixture: 1.6 ´ 10-7 M transcription ER expression gene 4-NP Straight chain: >10- activity Concentration: 5 M NP mixture: 10-11 - 10-5 M 4-NP mixture: (1/16,000 of that of 1.6 ´ 10-7 M E2) (E2: <10－11 M) 4-NP Straight chain: (1/1,000,000 or less of that of E2) 4-NP mixture: (1/16,000 or less of that of E2) Cells: HeLa cells transfected with PC10: NP mixture: Yamazaki et ER response sequence and human NP mixture: 6.0 ´ 10-4 M Positive in gene al., 2002 ER expression gene (E2: <10－9 M) transcription Concentration: 4-NP Straight chain: 10-11 activity (1/600,000 -11 -5 10 - 10 M - 10-5 M no effect or less of that of E2) 4-NP mixture: no effect

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Item Test methods and conditions Result Remark References Experiment that investigated the The potency of NP to No effect in gene Jorgensen, effect on the endogenous estrogen induce pS2 and TGFb3 expression 2000 responsive gene expression level gene was similar to that (the expression level of pS2, of genistein (GS) TGF b3, monoamine oxidase A whereas its action to (MAO-A), induce MAO-A was 10 a1-antichemotrypsin (a1-Act) was times higher than that of quantitated by PCR). GS but the induction of

a1-ACT was lower than that observed with GS. Alteration of Cells: Human breast cancer cells Cell growth activity was Positive in cell Soto et al., gene (MCF-7 cells, E-SCREEN assay) noted in the human growth activity 1991 expression serum preserved in a polystyrene tube, indicating that the activity was attributable to 4-NP extracted from the tube. Furthermore, progesterone receptor induction was also observed in the same experiment system. Human breast Cells: Human breast cancer cells The growth of MCF-7 Positive of cell White et al., cancer cell （MCF-7 and ZR-75 cells ） was observed after growth activity 1994 growth assay addition of 4-NP at > 10-5 M. Weak growth activity was noted with ZR-75. ER: Estrogen receptor; E2: 17b-estradiol; REC10: Concentration corresponding to 10% of 10-7 M E2 activity level; PC50: Concentration corresponding to 50% of the maximum activity by E2; IC50: 50% inhibition concentration; RBA: Relative bind strength (%)

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Attachment-2 Results of reproductivity and reproductive toxicity test in mammals

Species Route Period Dose Result References Rat p.o. For 3 days 4-NP (> 85%, Increased uterus weight at Laws et al., (Long (uterotro The uterus was branched mixture) 50 mg/kg/day or more 2000 Evans, phic dissected out at 0, 25, 50, 100, 200 female) assay) 6 hours after mg/kg/day 21- day old s.c. administration Increased uterus weight at (uterus and weighed 100 mg/kg/day or more weight assay) Rat p.o. 4-NP (> 85%, Increased uterus weight at (Long (uterotro branched mixture) 100 mg/kg/day or more Evans, phic 0, 25, 50, 100 female) assay) mg/kg/day 60-day old (ovariectomy 3 weeks before) Rat p.o. 3 days 4-NP（branched Increased uterus weight at Odum et al., (Alpk, (uterotro The uterus was mixture） > 75 mg/kg/day or more 1999 female) phic dissected out at 0, 37.5, 75, 150, 22 - 23-day assay) 24 hours after 225 mg/kg/day old administration 4-NP (branched Increased uterus weight at and weighed mixture) 250 mg/kg/day 0, 250 mg/kg/day Rat Increased uterus weight at (SD, 250 mg/kg/day female) 22 - 23-day old Rat 11 days 4-NP (branched Increased uterus weight at (Alpk, The uterus was mixture) 100 mg/kg/day female) dissected out at 0, 100 mg/kg/day 6 - 7 weeks 24 hours after old administration (ovariectom and weighed y when they s.c. For 11 days 4-NP（branched No increase in uterus weight was were 4 - 5 (uterotro Continuous mixture） observed in any of the groups weeks old) phic administration 0, 0.037, 27.2 assay) into mg/kg/day subcutaneously implanted mini- pump Rat p.o. 11 days 4-NP (branched Increased mammary gland lobule (Alpk, mixture) and S-stage cell count in the lobule female) 0, 100 mg/kg/day at 100 mg/kg/day 5 - 6 weeks s.c. 11 days 4-NP (branched No effect on the uterus and old Continuous mixture) mammary gland was observed in any administration 0, 0.052, 37.4 of the groups into mg/kg/day subcutaneously implanted mini- pump

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Species Route Period Dose Result References Rat s.c. twice Extracted significant increase in mitotic index Soto et al., (SD, female) administration polystyrene tube of endometrium at > 20 mg/rat 1991 (no after 19 days ingredient (4-NP description and 20 days mixture) of age) from 0, 1-50 mg/rat ovariectomy Rat s.c. 3 days 0, 2, 20, 200 Increased uterus weight at Yamasaki et (SD, (uterotro mg/kg/day 200 mg/kg/day or more al., 2001 female) phic 20 days old assay)

Rat s.c. Day 1 - 5 of 4-NP (Tokyo Abnormal estrus cycle, decreased Nagao et al., (SD, (uterotro birth Kasei Kogyo) fertility, increased atretic follicles 2000 female) phic 0, 500 mg/kg/day and decreased yellow bodies in the Neonate rat assay) ovary Rat i.p. Single dose NP (American The uterus weight, protein content, Lee & Lee， (SD, Cyanamid) DNA content and peroxidase activity 1996 female) 0, 1, 2, 4 mg/rat all increased in correlation to the dose from 1 mg/rat, and these activities were 1/1,000 - 1/2000 of that of 17b-estradiol. NP (American No effect was noted on the uterus Cyanamid) weight, protein content, DNA 4 mg/rat content and peroxidase activity + ICI 182,780 (estrogen antagonist) 50 mg/rat Rat p.o. 10 weeks NP (Aldrich Atrophy of seminiferous tubule at De Jager et al., (SD, male) Chemical) 100 mg/kg/day or more. 1999a, 1999b 12 weeks 0, 100, 250, 400 Decreased epididymis weight at old mg/kg/day 250 mg/kg/day. Decreased testis weight and number of sperm at 400 mg/kg/day Rat s.c. Day 1 - 5 of 4-NP (Tokyo Decreased germ cells in the Nagao et al., (SD, male) birth Kasei Kogyo) seminiferous tubule but no influence 2000 Neonate rat 0, 500 mg/kg/day on the motility of sperm and plasma testosterone concentration Rat i.p. Day 1 - 10 of 4-NP (branched No effect in the epididymis, prostatic Odum et al., (Alpk, birth mixture) ventral lobe and testis weights 2000 male） 0, 8 mg/kg/day Neonate rat Rat i.p. Day 1 - 15 of NP (American Decreased weights of the Lee，1998 (SD, male） birth Cyanamid) epididymis, prostatic ventral lobe Neonate rat 0, 0.08, 0.8, 8 and testis at 0.8 mg/kg/day and mg/kg/day above Curtailed AGD at 8 mg/kg/day i.p. Day 1 - 18 of NP (American Decreased weights of testis, birth Cyanamid) epididymis, seminal vesicle and 8 mg/kg/day prostatic ventral lobe were noted

322 Nonylphenol

Species Route Period Dose Result References Day 6 - 24 of Decreased weights of testis, Lee，1998 birth epididymis, seminal vesicle and prostatic ventral lobe Day 13 - 30 of No effect birth i.p. Day 1 - 5 of NP (American Decreased weights of testis, birth Cyanamid) epididymis, seminal vesicle and 8 mg/kg/day nondescended testes with atrophia (33.3%)

NP 8 mg/kg/day No effect on weights of testis, + epididymis, seminal vesicle ICI 182,780 (estrogen antagonist) 0.5 mg/kg/day i.p. Day 1 - 10 of NP (American Decreased weights of testis, birth Cyanamid) epididymis, seminal vesicle and 8 mg/kg/day nondescended testes with atrophy (54 - 62%)

NP 8 mg/kg/day No effect on weights of testis, + epididymis, seminal vesicle ICI 182,780 (estrogen antagonist) 0.5 mg/kg/day Rat By In utero, NP (Schenectady F0:Decreased in food consumption Ferguson et (SD, feeding lactation period International Inc.) in 25 ppm or more above al., 2000 female) 9- (soy- and immature 0, 25, 500, 2000 No effect on gestation period, birth

11/group bean Dams F0: GD 7 ppm weight, sex ratio, number of pups free) – PND 21 per litter F1: PND 21- F1:Decreased in body weight gain in 77d male 25 ppm or more and in female 2000 ppm Decreased in food consumption in male 2000 ppm group Increase in intake of water and saline water in both male and female 2000 ppm group

323 Nonylphenol

Species Route Period Dose Result References Rat By 4 generations 4-NP (mixture) at 200 ppm: No abnormality NTP, 1997; Chapin et al., (SD, female feeding 200, 650 and 650 ppm:, F2, F3: Decrease in body and male) 2,000 ppm weight increase, premature 1999 (corresponding to vaginal opening by 2 days

9-35, 30-100 and F1 females: Increased 100-350 uterus weight

mg/kg/day) F2: Decreased relative weight of ovary, decreased sperm concentration in the epididymis

2,000 ppm: F1, F2, F3:Decrease in body weight increase, premature vaginal opening by 6 days

F1 females: Increased uterus weight but no effect on the fertility

F2: Decreased relative weight of ovary, decreased sperm concentration in the epididymis, decreased spermatid count

Rat By F0 females: NP (American F1 males: Decreased sperm De Jager et al., (SD, female feeding During the Cyanamid) concentration in the epididymis at 1999a, 1999b and male) period of 0, 100, 250 250 mg/kg/day no descrip- pregnancy and mg/kg/day tion of age lactation up to 10 weeks after delivery

F0 males: For 10 weeks; F1: Up to 10 weeks after birth

324 Nonylphenol

Species Route Period Dose Result References

Rat p.o. F0 males: For 4-NP (Tokyo F0: Increased weights of liver, kidney Nagao et al, (SD, female (corn oil) 12 weeks before Kasei Kogyo) and pituitary, decreased weight of 2001 and male) mating 0, 2, 10, 50 thymus, increased serum TSH

25 rats/sex/ F0 females: For mg/kg/day (thyroid gland stimulating hormone) group 2 weeks before concentration in the males at 50 mating mg/kg/day; Decreased ovary weight maximum 2 in the females at 50 mg/kg/day

weeks during Decreased F1 survival rate in the the mating males and females at 50 mg/kg/day

period. F0 (day 0 and 4 of birth) males were NOAEL: 50 mg/kg/day autopsied after

mating, F1: Increased weights of liver and administration kidney, increased serum FSH was conducted (follicle stimulating hormone)

in F0 females concentration and decreased T3 throughout the (triiodothyronine) concentration (day period of 22 of birth) in the males at 50 pregnancy and mg/kg/day; Decreased ovary weight, lactation, and premature vaginal opening, they were decreased LH (luteinizing hormone) autopsied after and TSH concentration, and

weaning of F1. increased T3 concentration (day 22 Administration of birth) in the females at 50

to F1 rats after mg/kg/day weaning, Decreased number of implantations

mating in the and number of surviving F2 offspring same dose in the males and females at 50 group. The mg/kg/day

autopsy of F1 NOAEL: 10 mg/kg/day males and females were the same as that

of F0

325 Nonylphenol

Attachment-3 Repeated-dose toxicity test results

Species Route Period Dose Result References Rat gavage 28 days NP (Mitsui Toatsu Co., Increased relative weight of MHW, 1996 (SD, p.o. Ltd.) liver in the males at 60 female 0, 4, 15, 60, 250 mg/kg/day and male) mg/kg/day Salivation, suppression of 6 weeks body weight increase, old increased urine volume, decreased urine specific gravity, increased relative and absolute weights of liver, dilation of cecum, centrilobular hypertrophy of hepatocyte, basophilic change in the proximal tubule in the corticomedullary junction of kidney, basophilic change and dilation of collecting duct, hyperplasia of transitional epithelium of bladder in the males and females at 250 mg/kg/day Increased urea nitrogen and inorganic phosphorus, decreased chlorine, increased relative and absolute weights of kidney in the males at 250 mg/kg/day Scattering white spots in the kidney, swelling of kidney, dilation of renal pelvis, monocyte necrosis of epithelial cells of proximal tubule in the corticomedullary junction, inflammatory cell infiltration into interstitium, urinary cast, hyperplasia of the mucosa and dilation of renal pelvis in the females at 250 mg/kg/day Male: NOEL=15 mg/kg/day Female: NOEL= 60 mg/kg/day

326 Nonylphenol

Species Route Period Dose Result References Rat By 90 days NP (Schenectady Increased kidney weight Cunny et al., (SD, feeding International Inc.） (but returned to the normal 1997 female 0, 200, 650, 2,000 ppm level after 4 weeks of and male) (Corresponding to 0, 15, recovery period) and 6 weeks 50, 150 mg/kg/day) decreased hyaline droplets old in the tubular epithelium of kidney in the males at 2,000 ppm but nothing abnormal was noted in the estrus cycle and sperm test. NOAEL= 650 ppm (corresponding to 50 mg/kg/day)

327