NSF International has evaluated the noncancer oral toxicity data for p-tert-butylphenol, and developed an oral reference dose (RfD) based on a NOAEL of 70 mg/kg-day for decreased litter size and pup weights in F1 and F2 offspring, reduced body weight gain in both sexes of F0 parents, and reduced relative weight of ovaries and vaginal epithelial atrophy in F0 females observed in a two-generation reproduction study in rats (Clubb and Jardine, 2006). Applying an uncertainty factor of 1000 (10 each for inter- and intra-species variability; 3 each for extrapolation from a subchronic study and database deficiencies), resulted in an RfD of 0.07 mg/kg-day.

Noncancer Oral Specifics:

NSF Intl

DCE:
The endocrine activity of p-tert-butylphenol has been evaluated in several in vitro studies. On the basis of these assays, p-tert-butylphenol was shown to have intrinsic estrogenic activity, by virtue of its ability to bind the estrogen receptor (Soto et al., 1995; Routledge and Sumpter, 1997). The ability of other alkylphenols (e.g., p-nonylphenol) and phytoestrogens to mimic the effects of natural steroid hormones and their potential to disrupt the endocrine system in animals is an area of active investigation. The working definition of an endocrine disrupter adopted by U.S. EPA (1997) is "an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body which are responsible for the maintenance or homeostasis, reproduction, development and or behavior." As defined by the European Commission, an endocrine disruptor is "an exogenous substance that causes adverse health effects in an intact organism or its progeny through alterations in the function of the endocrine system" (EU, 2008). The data for p-tert-butylphenol from in vitro assays suggest that the relative potency of p-tert-butylphenol compared with the endogenous hormone, estradiol, is weak (over 1 million-fold lower), and thus unlikely to interfere with hormone binding. In vivo assays, however, are most relevant for predicting the potential for endocrine disruption.

Studies conducted according to the Organisation for Economic Co-operation and Development (OECD) Technical Guidelines 422 and 416 are considered the most relevant for detecting hormone-like effects of chemicals in vivo (Greim, 2004). OECD TG 422 is essentially a 6-week repeated-exposure study in conjunction with a reproductive/ developmental toxicity screen. By including the determination of organ weight and histopathology of hormone regulated organs, analysis of reproductive performance, and the development of offspring, it serves as an in vivo screen for the detection of endocrine effects. The NIHS (1996a) repeated dose reproductive/developmental screening study followed the OECD TG 422 to evaluate p-tert-butylphenol. There were no reproductive, developmental or systemic parental effects observed in this screening study with p-tert-butylphenol up to the highest dose of 200 mg/kg-day, although the presence of stridor observed in 4/13 females suggests that a maximally tolerated dose was attained.

The two-generation study (OECD TG 416) is currently the most complete protocol available for the evaluation of endocrine disrupters. The endocrine-disrupting potency of p-tert-butylphenol was assessed in vivo by a 2-generation reproduction study in rats, according to the OECD Guideline 416 (Clubb and Jardine, 2006). This test permits an in-depth study of the growth, development, and sexual function of the F1 generation and includes the monitoring of the subsequent F2 generation through weaning. The protocol includes parameters such as weights and pathologic examinations of the reproductive organs, detailed spermatogenesis, and sperm investigations of the parental generation and their offspring. Physical, sexual, and behavioral development and the learning and memory abilities of the offspring are also assessed (Greim, 2004). In the two generation reproduction study in rats, body weights or body weight gain was primarily affected in the first generation parents and offspring. Also affected in the F0 females were relative ovary weights and atrophy of the vaginal epithelium, however, these changes were not associated with altered sexual or reproductive performance under the conditions of the study.

QEST:
The two generation reproduction study in rats (Clubb and Jardine, 2006) was selected as the key study. The study was conducted according to current U.S. EPA health effects testing guidelines and OECD test guidelines. Decreased litter size and pup weights in F1 and F2 offspring, reduced body weight gain in both sexes of the F0 parents, and reduced relative ovary weights and vaginal epithelial atrophy in F0 females, were the critical effects, observed at the NOAEL of 70 mg/kg-day. The traditional NOAEL/UF approach was selected to derive the oral RfD using the uncertainty factors described here.

There are no directly comparable data in animals and humans from which to derive an interspecies uncertainty factor for of p-tert-butylphenol, thus, the default interspecies uncertainty factor of 10x based on administered dose was chosen. Due to the lack of kinetics or metabolism data for p-tert-butylphenol in humans, a 10x uncertainty factor was considered appropriate to account for potential intraspecies differences in toxicokinetics or toxicodynamics. A LOAEL to NOAEL extrapolation uncertainty factor was not applied, since the key study identified a NOAEL (Clubb and Jardine, 2006).

Studies in rats conducted by the oral route showed similar body weight reductions at comparable dose levels when given for subchronic (16 weeks) and chronic (51 weeks) periods, suggesting that the longer exposure duration did not exacerbate the effect on body weight. Thus in the two generation rat reproduction study (Clubb and Jardine, 2006), the mean body weight of male rats given 600 mg/kg-day for 16 weeks was decreased 16\% relative to controls. A 16\% reduction in body weight was also observed male rats given ~750 mg/kg-day in the 51 week study (Hirose et al., 1988). Body weight is a relevant endpoint because it is among the critical effects identified in the two-generation reproduction study in rats. The magnitude of effect on body weight is similar whether the dosing period was 16 weeks or 51 weeks, suggesting a 1x uncertainty factor may be appropriate. In the absence of data to permit quantitative extrapolation of all subchronic critical effects to chronic exposure, and in the absence of a study of full two-year duration, a subchronic to chronic extrapolation uncertainty factor of 3x was chosen.

The database does not include a 90-day study; however, this deficiency can be met by the two-generation rat reproduction study, which is a repeated dose oral study with exposure duration of greater than 90 days. The database also lacks developmental toxicity studies in two species; however, the two-generation reproduction study in rats permits a full assessment of the reproductive process, including developmentally induced effects on the reproductive systems of offspring. Additionally, due to the negative results in a combined repeat dose reproductive/developmental toxicity screening test, the full developmental study in rats was not required for this evaluation. The weight of evidence suggests that this chemical is not genotoxic in vivo. While studies of full two-year duration are not available, p-tert-butylphenol and other members of the alkylphenol class have been characterized as possible tumor promoters based on forestomach lesions in repeated dose oral studies in hamsters (20 weeks) and rats (51 weeks) and its promoting activity in an initiation-promotion assay with N-methyl-N'-nitro-N-nitrosoguanidine. Tumor development in the forestomach has been associated with chronic tissue irritation (IARC, 2003). A 3x database uncertainty factor was considered appropriate due to the availability of a two-generation reproduction study, chronic studies with limited endpoints in two species, and a developmental toxicity screening test in rats, but no developmental toxicity assessment in a second species. The total uncertainty factor is, therefore, 1000x.

The oral RfD is based on the NOAEL of 70 mg/kg-day from the two-generation reproduction study in rats (Clubb and Jardine, 2006). Applying an uncertainty factor of 1000 results in an oral RfD of 0.07 mg/kg-day.

PEER:
This NSF International oral risk assessment document was externally peer reviewed by the following NSF Health Advisory Board Members: E. Ohanian, U.S. Environmental Protection Agency; M. Dourson, TERA (Toxicology Excellence for Risk Assessment); D. Blakey, Health Canada; S. Bursian, Michigan State University; C. Farr, Consultant; R. Hinderer, The Lubrizol Corporation; E. McConnell, ToxPath, Inc.; J. Orme-Zavaleta, U.S. Environmental Protection Agency; and C. Willhite, Department of Toxic Substances Control, State of California.

BIB:
Clubb, S. and L. Jardine. 2006. p-tert-Butylphenol two generation reproduction study in rats. Charles River Laboratories Study Number 493595. EPA-HQ-OPPT-2006-0961-0032[1].

EU (European Union). 2008. European Union Risk Assessment Report. P-TERT-BUTYLPHENOL; CAS No: 98-54-4; EINECS No: 202-679-0. Final Report, 2008. Norwegian Pollution Control Authority, R404 1208ENV\_HH.

Greim, H.A. 2004. The Endocrine and Reproductive System: Adverse Effects of Hormonally Active Substances? Pediatrics. 113(4): 1070-1075.

Hirose, M., S. Fukushima, Y. Kurata, H. Tsuda, M. Tatematsu, and N. Ito. 1988. Modification of n methyl-n'-nitro-n-nitrosoguanidine-induced forestomach and glandular stomach carcinogenesis by phenolic antioxidants in rats. Cancer Res 48(18): 5310-5315.

IARC (International Agency for Research on Cancer). 2003. Predictive Value of Rodent Forestomach and Gastric Neuroendocrine Tumors in Evaluating Carcinogenic Risk to Humans. International Agency for Research on Cancer (IARC), Lyon, France. IARC Technical Publication No. 39.

NIHS (National Institute of Health Sciences), Japan. 1996a. Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test of p-tert-butylphenol by Oral Administration in Rats. Hatano Research Institute. Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Available online at: http://dra4.nihs.go.jp/mhlw\_data/home/paper/paper98-54-4d.html

Routledge, E. J. and J. P. Sumpter. 1997. Structural features of alkylphenolic chemicals associated with estrogenic activity. Journal of Biological Chemistry 272(6): 3280-3288.

Soto, A.M., C. Sonnenschein, K.L. Chung, M.F. Fernandez, N. Olea, and F. Olea Serrano. 1995. The E-SCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environmental Health Perspectives 103(SUPPL. 7): 113-122.

U.S. EPA (U.S. Environmental Protection Agency). 1997. Special report on Environmental Endocrine Disruption: An Effects Assessment and Analysis. Office of Research and Development, EPA/630/R-96/012, Washington D.C.

MOREI:
NSF International. 2010. P-Tert-Butylphenol. CAS# 98-54-4. Oral Risk Assessment Document. December. Available for a fee at http://www.techstreet.com/standards/NSF/P\_Tert\_Butylphenol\_2010?product\_id=1763966

An Executive Summary of the NSF International oral risk assessment document for p-tert-butylphenol is available at http://www.techstreet.com/direct/nsf/P-Tert-Butylphenol\_es.pdf