Hypothesis-driven weight-of-evidence analysis of endocrine disruption potential: a case study with triclosan

Figures & data

Figure 1. Chemical structure of triclosan.

Table 1. Ranked endpoints for relevant triclosan data for the estrogen agonist hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	Fish Short-Term Reproduction Assay	Vitellogenin: increased in males		{1} [2] [3]
	Uterotrophic assay	Uterine weight	No effect	[4] [5]
2	ERTA	ER agonism	No effect	[5] [6]
	Fish Short-Term Reproduction Assay	2° sex characteristics: tubercle score	No effect	[2] [3]
		Gonad histopathology	No effect	[3]
		Behavior	No effect	[3]
	Pubertal female assay	Age & weight @ vaginal opening		{4}
		Ovary weight	No effect	[4]
		Age @ 1st estrous	No effect	[4]
	Pubertal male assay	Testes weight	No effect	[7]
		Testes histopathology: atrophy	No effect	[7]
3	ER binding	ER competitive binding	–	–
	Fish Short-Term Reproduction Assay	Fecundity	No effect	[1] [2]
		Behavior	–	–
		Gonadosomatic Index	No effect	[2]
		Gonadosomatic Index		{1} [2]
		Gonad histopathology: follicular atresia	No effect	[3]
		Fertilization success	No effect	[1] [2]
		Estradiol	No effect	[2]
		Testosterone	No effect	[2]
	Pubertal female assay	Growth	No effect	[4]
		Uterus weight		{4}
		Estrous cyclicity	–	–
	Pubertal male assay	Growth	No effect	[7]
		Ventral prostrate weight	No effect	[7]
		Epididymis histopathology	No effect	[7]
	Steroidogenesis assay	Estradiol levels	–	–

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses are considered equivocally reliable. A line indicates that data is not available for that endpoint.

[1] Ishibashi et al. (2004).

[2] BASF (2012).

[3] Schultz et al. (2012).

[4] Stoker et al. (2010).

[5] Louis et al. (2013).

[6] Ahn et al. (2008).

[7] Zorrilla et al. (2009).

Table 2. Ranked endpoints for relevant triclosan data for the estrogen antagonist hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	No Rank 1 endpoints identified by Borgert et al. (2014)
2	Pubertal female assay	Age & weight @ vaginal opening	No effect	[1] [2]
		Age @ 1st estrous	No effect	[1] [2]
	Fish Short-Term Reproduction Assay	Vitellogenin: reduced in females	No effect	[3] [4] [5]
		Gonad histopathology	No effect	[5]
3	Aromatase assay	Aromatase Assay	–	–
	Steroidogenesis assay	Steroidogenesis Assay	–	–
	Pubertal female assay	Estrous cyclicity	–	–
		Ovary histopathology: atrophy	–	–
		Ovary weight (with atrophy)	No effect	[1]
		Uterus weight	No effect*	[1]
	Fish Short-Term Reproduction Assay	Fecundity	No effect	[3] [4]
		Gonadosomatic Index		{3} [4]
		Behavior	No effect	[5]
		Fertilization success	No effect	[3] [4]
		Estradiol	No effect	[4]
		Testosterone	No effect	[4]

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses are considered equivocally reliable. A line indicates that data is not available for that endpoint.

* Uterine weight increased in the study by Stoker et al. (2010), however, an estrogen antagonist would be expected to decrease uterine weight so this endpoint is listed as “no effect” for the estrogen antagonist hypothesis.

[1] Stoker et al. (2010).

[2] Louis et al. (2013).

[3] Ishibashi et al. (2004).

[4] BASF (2012).

[5] Schultz et al. (2012).

Table 3. Ranked endpoints for relevant triclosan data for the androgen agonist hypothesis.

Rank	Assay	Endpoint(s)	Response		Reference(s)
1	Hershberger	Weights of five androgen-dependent tissues	–		–
	Fish Short-Term Reproduction Assay	2° sex characteristics: tubercle score	No effect		[1] [4]
2	Pubertal male assay	Age & weight @ preputial separation	No effect		[2]
		Seminal vesicle + coagulating gland weight	No effect		[2]
		Ventral prostate weight	No effect		[2]
		Dorsolateral prostate weight	–		–
		LABC muscle complex weight	No effect		[2]
		Epididymis weight	No effect		[2]
		Testes weight	No effect		[2]
		Testes histopathology	No effect		[2]
		Epididymis histopathology	No effect		[2]
	Fish Short-Term Reproduction Assay	Vitellogenin	No effect		[1] [3] [4]
		Gonad histopathology	No effect		[4]
	AR binding	Competitive binding	–		–
	Hershberger	Concordance of two or more endpoints	–		–
3	Aromatase assay	Aromatase activity	–		–
	Fish Short-Term Reproduction Assay	Fecundity	No effect		[1] [3]
		Testosterone and estradiol	No effect		[1]
		Gonadosomatic Index			[1] {3}
		Behavior	No effect		[4]
		Fertilization success	No effect		[1] [3]
	Pubertal female assay	Growth	No effect		[5]
		Age & weight @ vaginal opening			{5}
		Uterine weight			{5}
		Ovaries weight	No effect		[5]
		Adrenals weight	–		–
		Uterus histopathology	No effect		[5]
		Ovary histopathology	–		–
	Pubertal male assay	Growth	No effect		[2]
		Serum testosterone	No effect		[2]
	Steroidogenesis assay	Testosterone levels	–		–
	Hershberger	Only one of five endpoints respond	–		–

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses are considered equivocally reliable. A line indicates that data is not available for that endpoint.

[1] BASF (2012).

[2] Zorrilla et al. (2009).

[3] Ishibashi et al. (2004).

[4] Schultz et al. (2012).

[5] Stoker et al. (2010).

Table 4. Ranked endpoints for relevant triclosan data for the androgen antagonist hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	Hershberger	Weights of five androgen-dependent tissues	–	–
2	Pubertal male assay	Age & weight @ preputial separation	No effect	[1]
		Seminal vesicle + coagulating gland weight	No effect	[1]
		Ventral prostate weight	No effect	[1]
		Dorsolateral prostate weight	–	–
		LABC muscle complex weight	No effect	[1]
		Epididymis weight	No effect	[1]
		Testes weight	No effect	[1]
		Testes histopathology	No effect	[1]
		Epididymis histopathology	No effect	[1]
	Fish Short-Term Reproduction Assay	Vitellogenin	No effect	[2] [3] [4]
		2° sex characteristics: tubercle score	No effect	[3] [4]
		Gonad histopathology	No effect	[4]
	AR binding	Competitive binding	–	–
	Hershberger	Concordance of two or more endpoints	–	–
3	Steroidogenesis assay	Testosterone levels	–	–
	Fish Short-Term Reproduction Assay	Fecundity	No effect	[2] [3]
		Testosterone and estradiol	No effect	[3]
		Gonadosomatic index		{2} [3]
		Behavior	No effect	[4]
		Fertilization success	No effect	[2] [3]
	Pubertal male	Serum testosterone	No effect	[1]
	Hershberger	Only one of five endpoints respond	–	–

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses are considered equivocally reliable. A line indicates that data is not available for that endpoint.

[1] Zorrilla et al. (2009).

[2] Ishibashi et al. (2004).

[3] BASF (2012).

[4] Schultz et al. (2012).

Table 5. Ranked endpoints for relevant triclosan data for the thyroid agonist hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	Amphibian metamorphosis assay	Asynchronous development	No effect	[1] [2]
		Thyroid histopathology	No effect	[1] [2]
2	Amphibian metamorphosis assay	Developmental stage	No effect	[1] [2]
		Hind limb length	No effect	[1] [2]
	Pubertal male assay	Thyroid weight	–	–
		Serum T4		{3}
		Serum TSH	No effect	[3]
	Pubertal female assay	Serum T4		{4}
		Serum TSH	No effect	[4]
3	Amphibian metamorphosis assay	Snout-vent length	No effect*	[1] [2]
		Wet weight	No effect*	[1] [2]
	Pubertal female assay	Growth	No effect	[4]
		Age & weight @ vaginal opening		{4}
		Blood chemistry	–	–
	Pubertal male assay	Growth	No effect	[3]
		Age & weight @ preputial separation	No effect	[3]
		Pituitary weight	No effect	[3]

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses denotes equivocally reliable. A line indicates that data is not available for that endpoint.

* Snout-vent length and wet weight were reported to have increased in the study by Fort et al. (2011), however, a thyroid agonist would be expected to decrease these parameters so they are shown as “no effect” for this hypothesis.

[1] Fort et al. (2010).

[2] Fort et al. (2011).

[3] Zorrilla et al. (2009).

[4] Stoker et al. (2010).

Table 6. Ranked endpoints for relevant triclosan data for the thyroid antagonist hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	Amphibian metamorphosis assay	Asynchronous development	No effect	[1] [2]
		Thyroid histopathology	No effect	[1] [2]
	Pubertal female Assay	Thyroid weight	–	–
		Thyroid histopathology	–	–
	Pubertal male assay	Thyroid weight	–	–
		Thyroid histopathology		{3}
2	Pubertal male assay	Liver weight		{3}
		Serum T4		{3}
		Serum TSH	No effect	[3]
	Pubertal female assay	Age & weight @ vaginal opening		{4}
		Ovary weight	No effect	[4]
		Serum T4		{4}
		Serum TSH	No effect	[4]
3	Pubertal male assay	Growth	No effect	[3]
		Age & weight @ preputial separation	No effect	[3]
		Pituitary weight	No effect	[3]
	Pubertal female assay	Growth	No effect	[4]
		Blood chemistry	–	–
	Amphibian metamorphosis assay	Development	No effect	[1] [2]
		Snout-vent length		[1] {2}
		Hind limb length	No effect	[1] [2]
		Wet weight		[1] {2}

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses denotes equivocally reliable. A line indicates that data is not available for that endpoint.

[1] Fort et al. (2010).

[2] Fort et al. (2011).

[3] Zorrilla et al. (2009).

[4] Stoker et al. (2010).

Table 7. Ranked endpoints for relevant triclosan data for the steroidogenesis induction hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	No Rank 1 endpoints identified by Borgert et al. (2014)
2	Steroidogenesis assay	Estradiol levels	–	–
		Testosterone levels	No effect	[1]
3	Male pubertal assay	Serum testosterone levels	No effect	[2]

Endpoint responses compared to controls for each study are noted. A line indicates that data is not available for that endpoint.

[1] Forgacs et al. (2012).

[2] Zorrilla et al. (2009).

Table 8. Ranked endpoints for relevant triclosan data for the steroidogenesis inhibition hypothesis.

Rank	Assay	Endpoint(s)	Response	Reference(s)
1	Pubertal female	Uterus weight	No effect*	[1]
2	Steroidogenesis	Estradiol levels	–	–
		Testosterone levels	No effect	[2]
	Pubertal female	Ovary weight	No effect	[1]
	Fish Short-Term Reproduction Assay	Vitellogenin	No effect	[3] [4] [5]
		Gonad histopathology	No effect	[5]
3	Aromatase assay	Aromatase activity	–	–
	Fish Short-Term Reproduction Assay	Fecundity	No effect	[3] [4]
		Behavior	No effect	[5]
		Gonadosomatic index		{3} [4]
		Fertilization success	No effect	[3] [4]
		Estradiol	No effect	[4]
		Testosterone	No effect	[4]
	Pubertal female assay	Age @ 1st estrous	No effect	[1]
		Age & weight @ vaginal opening	No effect	[1]

Endpoint responses compared to controls for each study are noted. Studies with a response for an endpoint are noted in {bold}. Shaded endpoint responses are considered equivocally reliable. A line indicates that data is not available for that endpoint.

* Uterine weight increased in the study by Stoker et al. (2010), however, an aromatase inhibitor would be expected to decrease uterine weight so this endpoint is listed as “no effect” for the steroidogenesis inhibition hypothesis.

[1] Stoker et al. (2010).

[2] Forgacs et al. (2012).

[3] Ishibashi et al. (2004).

[4] BASF (2012).

[5] Schultz et al. (2012).

Figure 2. ToxCast concentration distribution for assays active for triclosan. The endocrine-specific receptor assays are estrogen (ER), androgen (AR), thyroid (ThR), and aromatase. The solid line represents the median cytotoxicity limit and the dashed line is the lower bound (5%) cytotoxicity limit.

Table 9. Histopathological examination of thyroid in repeat dose studies with various species, duration, and route of exposure to triclosan.

Study type	Species	Doses
One year oral toxicity study*	Baboon	0, 30, 100, and 300 mg/kg
Two year oral (feed)*,†	Rat	0, 300, 1000, 3000 (MTD), 6000 (Toxic) ppm
90 day Bathing study (TSC soap solution)*	Rhesus Monkeys	Bathed 5 min daily with soap solution containing 1% TCS (no untreated controls but controls bathed in –TCS soap included)
Carcinogenicity study (diet, 95 weeks)†	Hamsters	0, 12.5, 75, 250 mg/kg/day
90 day oral tox, (feed)*	Rabbits	0, 250, 500, 1250, 2500 ppm
90 day oral tox (feed)*	Dog	0, 125, 313, 625 ppm (0, 5, 12.5, 25 mg/kg)
90 day oral (diet)*,†	Rats	0, 1000, 3000, 6000 ppm
4 and 13 week oral tox*,†	Baboon	0, 1, 3, 10, 30, 100 mg/kg/day (4 weeks), 13 weeks 3 mg/kg/day
18-month oral tox*,†	Mouse	0, 10, 30, 100, 200 mg/kg/day
90 day dermal tox†	Rat	0, 10, 40, 80 mg/kg/day

In each of the studies identified in the public domain (ECHA 2015; SCCP 2009) the summary reports indicate that no histopathological changes to thyroid have been observed in the triclosan-treated groups compared to control animals.

* European Commission, Scientific Committee on Consumer Safety; Opinion on Triclosan, 2009.

† Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulation, dissemination portal of the European Chemicals Agency (ECHA). Data accessed on Triclosan (CAS 3380–34-5).

Ishibashi H, Matsumura N, Hirano M, Matsuoka M, Shiratsuchi H, Ishibashi Y, Takao Y, Arizono K. 2004. Effects of triclosan on the early life stages and reproduction of medaka Oryzias latipes and induction of hepatic vitellogenin. Aquat Toxicol. 67:167–179.

 PubMed Web of Science ®Google Scholar

BASF. 2012. Robust study summary: evaluation of reproductive toxicity to fathead minnow (Pimephales promelas) using 21-d fish reproduction screen (EPA/600/R-01/067) [Internet]. [cited 2015 Dec 18]. In: ECHA 2015, Triclosan REACH dossier. Available from: http://echa.europa.eu/information-on-chemicals/registered-substances.

 Google Scholar

Schultz MM, Bartell SE, Schoenfuss HL. 2012. Effects of triclosan and triclocarban, two ubiquitous environmental contaminants, on anatomy, physiology, and behavior of the fathead minnow (Pimephales promelas). Arch Environ Contam Toxicol. 63:114–124.

 PubMed Web of Science ®Google Scholar

Stoker TE, Gibson EK, Zorrilla LM. 2010. Triclosan exposure modulates estrogen-dependent responses in the female wistar rat. Toxicol Sci. 117:45–53.

 PubMed Web of Science ®Google Scholar

Louis GW, Hallinger DR, Stoker TE. 2013. The effect of triclosan on the uterotrophic response to extended doses of ethinyl estradiol in the weanling rat. Reprod Toxicol. 36:71–77.

 PubMed Web of Science ®Google Scholar

Ahn KC, Zhao B, Chen J, Cherednichenko G, Sanmarti E, Denison MS, Lasley B, Pessah IN, Kultz D, Chang DP, et al. 2008. In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens. Environ Health Perspect. 116:1203–1210.

 PubMed Web of Science ®Google Scholar

Zorrilla LM, Gibson EK, Jeffay SC, Crofton KM, Setzer WR, Cooper RL, Stoker TE. 2009. The effects of triclosan on puberty and thyroid hormones in male Wistar rats. Toxicol Sci. 107:56–64.

 PubMed Web of Science ®Google Scholar

Borgert CJ, Stuchal LD, Mihaich EM, Becker RA, Bentley KS, Brausch JM, Coady K, Geter DR, Gordon E, Guiney PD, et al. 2014. Relevance weighting of tier 1 endocrine screening endpoints by rank order. Birth Defects Res B Dev Reprod Toxicol. 101:90–113.

 PubMed Web of Science ®Google Scholar

Fort DJ, Mathis MB, Hanson W, Fort CE, Navarro LT, Peter R, Buche C, Unger S, Pawlowski S, Plautz JR. 2011. Triclosan and thyroid-mediated metamorphosis in anurans: differentiating growth effects from thyroid-driven metamorphosis in Xenopus laevis . Toxicol Sci. 121:292–302.

 PubMed Web of Science ®Google Scholar

Fort DJ, Rogers RL, Gorsuch JW, Navarro LT, Peter R, Plautz JR. 2010. Triclosan and anuran metamorphosis: no effect on thyroid-mediated metamorphosis in Xenopus laevis. Toxicol Sci. 113:392–400.

 PubMed Web of Science ®Google Scholar

Forgacs AL, Ding Q, Jaremba RG, Huhtaniemi IT, Rahman NA, Zacharewski TR. 2012. BLTK1 murine Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and developmental toxicants. Toxicol Sci. 127:391–402.

 PubMed Web of Science ®Google Scholar

ECHA [Internet]. 2015. Triclosan REACH dossier. [cited 2016 Feb 15]. Available from: http://echa.europa.eu/information-on-chemicals/registered-substances.

 Google Scholar

SCCP [Internet]. 2009. Scientific Committee on Consumer Products: Opinion on Triclosan. [cited 2016 Mar 15]. Available from: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_166.pdf, European Commission.

 Google Scholar