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Statement on potential endocrine activity of TCPP (EC No. 911-815-4) of October 2015

 

Background:

TCPP is selected as a compound for the REACH CoRAP process. A justification document for the selection is provided by the Danish Environmental Protection Agency (CoRAP Justification, 2015). In this justification initial grounds for concern to be clarified under substance evaluation are indicated including “potential endocrine disruptor” and the following justification is given:

“The endocrine disruption potential of TCPP was investigated in an in vitro study with the H295R cell line where testosterone concentration was increased at 1, 10 and 100 mg/L. Furthermore, data from the 2-generation reproductive toxicity study (described above) indicate hormonal disturbance by TCPP due to the findings concerning decreased uterus weight and also prolongation of the oestrus cycle. The results indicate that TCPP could alter the sex hormone balance. This could support a classification as indicated above. However, it remains to be determined whether increased testosterone level also occurs in vivo and whether this could be associated to the decrease in uterus weight. Thus, further verification/studies would be needed to clarify the potential for endocrine disruption of the substance.” (CoRAP Justification, 2015).

In this statement we summarize information on potential endocrine activity of TCPP taking into account in vitro and in vivo studies and evaluations by other expert groups.

 

Executive Summary:

Two in vivo effects are mentioned in the CoRAP justification document observed in a 2-generation reproductive toxicity study; decreased uterus weight and also prolongation of the oestrus cycle. As mentioned in the EU-RAR (2008) “It is noted that all organ weight changes occurred in the absence of any histopathological changes, and it is accepted that uterine weight can fluctuate during the oestrus cycle. Therefore, the effects observed may be due to normal variation in cycling females.” This interpretation is in agreement with the conclusions drawn by the study director and the observation may thus be judged as maternal toxicity but not as adverse with regard to fertility.

The majority of in vitro data investigating a potential endocrine activity of TCPP is negative. One study reported increased testosterone excretion in H295R cells at high concentrations at or above 1 mg/l and increased E2 excretion and mRNA expression of metabolic enzymes at even higher concentrations; in the same study zebrafish was also investigated and no effect on any of these parameters is reported in the fish model.

 

 

Overall, there is no convincing evidence that TCPP has endocrine activity.

 

Potential Read Across to Other Phosphate Esters:

No read across to other phosphate esters is taken into account in this statement based on the following evaluations:

The Danish Environmental Protection Agency evaluated TCPP in 2014 and concluded on a read across: “Read-across to TCEP in relation to the reproductive toxicity (as done for the carcinogenic effects) seems less reliable as no effects on uterus have been found for TCEP, and also TCEP strongly affect the male reproductive system which has not been found for TCPP.” (Danish EPA. 2014).

The US Department of Health and Human Services, Public Health Services, Agency for Toxic Substance and Disease Registry (US ATSDR) discussed a potential read across between phosphate esters as follows: “This profile discusses the following phosphate ester flame retardants: tris(2-chloroethyl) phosphate (TCEP), tributyl phosphate (TnBP), tris(2-butoxyethyl) phosphate (TBEP), tris(1,3-dichloro-2-propyl) phosphate (TDCP), triphenyl phosphate (TPP), tris(2-chloroisopropyl) phosphate (TCPP), triisobutyl phosphate (TiBP), and tricresyl phosphate (TCP).

… Endocrine parameters evaluated in the toxicity studies available generally consisted of the weight and gross and microscopic morphology of endocrine glands (i.e., thyroid, pituitary, adrenals). As discussed below, except for TCP, no significant alterations were reported in endocrine glands following oral exposure to the phosphate ester flame retardants discussed in this profile.” (US-ATSDR. 2012).

In the EU risk Assessment Report 2008 a qualitative (not a quantitative) read-across for the endpoint carcinogenicity (no other end point) is performed: “It is considered that there is sufficient information from the structures, physical chemical properties, toxicokinetics and mutagenic profiles of TCEP, TDCP and TCPP to support a qualitative read-across to address the hazard and risk assessment for the carcinogenicity endpoint for TCPP. However, it is accepted that there are some differences in the metabolism, the target organs and the severity of the effects observed with the three substances. Also, there are no insights into an underlying mode of action for TCEP and TDCP which would make a prediction on a relatively potency of TCPP possible. Therefore, a quantative read-across to carcinogenicity data of either TCEP or TDCP was not performed”…”No quantitative read-across can be performed since there are no insights into an underlying mode of action for TCEP and TDCP which would make a prediction on a relatively potency of TCPP possible. Therefore, as a reasonable worst case approach, a risk characterisation will be carried out for this end-point [carcinogenicity].” (EU-RAR. 2008)

 

In Vitro TCPP Studies:

A study by Liu et al (2012) is mentioned in the CoRAP justification by the Danish EPA (CoRAP justification. 2015).

Liu et al. (2012) investigated different phosphate esters including TCPP. Sex hormone synthesis and steroidogenic gene transciption is investigated in H295R cells. With MVLN cells, estrogen receptor binding activities are evaluated and in zebrafish, sex hormones and related gene transcriptions are determined. TCPP did not show cytotoxicity in H295R and MVLN cells even at 100 mg/L (data not shown). In MVLN cells, TCPP does not act as estrogen receptor agonists or antagonists. TCPP dependent increase in 17β-estradiol (E2) excretion into the medium is observed with H295R cells at 100 mg/l (no effect at 10 mg/l or lower concentrations); increased testosterone excretion at or above 1 mg/l (no effect at 0.1 mg/l or lower concentrations); increased CYP1A11 mRNA expression at 100 mg/l (no effect at 10 mg/l or lower concentrations); increased CYP 2B11 and HSD3β2 mRNA expression at 10 mg/l (no effect at 1 mg/l or lower concentrations) and no effect on CYP1A19 mRNA expression. Reduced SULT 1E1 and SULT2A1 m-RNA expression is observed at 10 mg/l (no effect at 1 mg/l or lower concentrations). In zebrafish, no effect is reported on concentrations of sex hormones E2, testosterone (T) or 11-ketoestrosterone (11-KT) in blood plasma, and gene transcription of CYP17 and CYP19A in gonad, and vitellogenin 1 in liver (Liu et al. 2012).

 

In Addition the Following in Vitro Studies are identified:

Föllmann and Wober. 2006: Estrogenic or anti-estogenic effects are examined with the recombinant yeast reporter gene assay, and in human endometrial cancer Ishikawa cells by induction of alkaline phosphatase. - Hormonal activity shown as induction of estrogenic or anit-estogenic effects could not be detected in the two in vitro test systems for TCPP.

Zhang et al. 2014: The agonistic/antagonistic activity of a group of phosphate esters is examined by three in vitro models (estrogen receptor (ER) dependent luciferase reporter gene assay, yeast two-hybrid assay, and E-screen assay). Molecule docking is used to further describe the potential interactions between ERα and the compounds investigated. TCPP had no estrogenic or anti-estrogenic activity in any of the tests performed.

Svenson et al. 2011: An vitro test based on recombinant yeast strains transfected with genes for the human estrogen receptor is reported to examine in vitro estrogenic and anti-estrogenic activity. TCPP is found inactive under all test conditions.

Kojima et al. 2013: Cell-based transactivation assays are reported to investigate potential agonistic and/or antagonistic activities of different phosphate esters against human nuclear receptors; estrogen receptor alpha (ER alpha), ER beta, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor alpha 1(TR alpha1), TR beta1,retinoic acid receptor alpha (RAR alpha), retinoid X receptor alpha (RXR alpha), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPAR alpha), and PPAR beta. Compounds are indicated as not active with RIC20 or REC20 value (concentration equal to 20% of the maximal response of the positive control) above 10 µM. TCPP is inactive in all tests with the exception of one test. A REC20 value of 4.9 ± 2µM is reported for PXR agonistic activity, close to the 10µM limit concentration.

Suzuki et al. 2013: CALUX Assays to test AR-, ERα-, PR-, GR-, and PPARg2-activity in human osteosarcoma cell lines that are stably co-transfected with individual target human-receptor-regulated luciferase gene constructs (U2OS-luc cells). No agonistic effects are reported for TCPP. Antagonistic activity is reported at high concentrations for AR (RIC20, 10 µM), PR (RIC20, 3 µM). Compounds are indicated as not active with REC20 value above 10 µM.

In conclusion, the majority of in vitro data are negative. Observations at high concentrations are reported in a few studies; in one study AR inhibition (10µM) and PR inhibition (3µM); in one study PXR agonistic activity at 4.9 µM. One study reported increased T excretion in H295R cells at 1 mg/l and increased E2 excretion and mRNA expression of metabolic enzymes at even higher concentrations; in the same study no effect on any of these parameters is reported in zebrafish. Studies on agonistic and/or antagonistic activities are reported to be negative for of ER alpha, ER beta, AR, GR, TR alpha1, TR beta1, RAR alpha, RXR alpha, PPAR alpha, and PPAR beta.

 

Toxicokinetic Data:

Toxicokinetic data on TCPP are summarized in the EU-RAR (2008) and blood and tissue Cmax concentrations are reported after IV and oral dosing of Sprague-Dawley rats. “Cmax after intravenous injection of 20 mg/kg averaged 142 μg equivalents/ml and was reached in 0.15 hours while after oral administration of 20 mg/kg a Cmax of 7.68 μg equivalents/ml and was reached in 0.5 hours. Cmax after high dose oral administration [200 mg/kg] was 84 μg equivalents/ml and was reached within 2 hours.” (EU-RAR 2008). The maximal in vivo serum or tissue concentrations (Cmax) demonstrate that the in vitro observation mentioned above at or above 3µM TCPP (corresponds to approx. 1µg/ml; TCPP MW 327) might be reached temporarily in rodents only at very high doses above 1 mg/kg.

 

In Vivo TCPP Studies:

Systemic toxicity:

In the sub-chronic study rats were fed diets containing 0, 800, 2500, 7500 and 20000 ppm of TCPP for a period of thirteen weeks. This corresponds to mean substance intake values of 0, 52, 160, 481, and 1349 mg/kg/day for males and 0, 62, 171, 570, and 1745 mg/kg/day for females. This study indicated the liver and thyroid to be the main target organs affected by TCPP. Effects observed included statistically significant increases in absolute and relative liver weights in males at all doses and females at the two highest doses, periportal hepatocyte swelling in high dose groups and mild thyroid follicular cell hyperplasia in males at all doses and females at the highest dose. Based on the increase in both absolute and relative liver weights, accompanied by mild thyroid follicular cell hyperplasia observed in males of all dose groups, a LOAEL of 52 mg/kg/day is derived (Staufer Chemical Company 1981). The presence of thyroid follicular cell hypertrophy is considered to be secondary to hepatocellular microsomal enzyme induction (NTP Nonneoplastic Lesion Atlas, 2015).

4-week oral gavage study with 0, 10, 100 and 1000 mg/kg/day also showed the liver as the target organ, with increased liver weight observed in the high dose groups, accompanied by hepatocyte hypertrophy in all high–dose males and one mid-dose male and changes in ALAT activity in high-dose animals (Leser.1991).

TCPP is currently within the testing procedure of the National Toxicology Program (NTP) with sub-chronic and chronic studies in rats and mice. Preliminary results obtained from the NTP webpage for a 13 week feeding study on male mice indicate a dose-dependently reduced body weight at termination and that liver and kidney are the main target organs affected by TCPP. Female mice seem to be less susceptible than male mice with a reduction in body weight and adverse liver effects at higher doses (NTP, 2015).

In a 2-generation reproductive toxicity study in which rats were fed TCPP in the diet over two successive generations 28 animals/sex/group received TCPP in the diet, corresponding approximately to 0, 85, 293 and 925 mg/kg/day for males and 0, 98.6, 329.9 and 988.2 mg/kg/day for females. The low-dose of 99 mg/kg/day for females is considered to be the LOAEL for parental toxicity. This is based on decreased body weight and food consumption seen in mid and high dose parental animals and liver weight increase in the high dosed females. For males, a NOAEL of approximately 85 mg/kg/day is derived for parental toxicity, based on decreased body weights, food consumption and organ weight changes observed at mid and high dose groups. The observed relative liver weight increase observed at all dose level is not regarded as adverse (Waalkens-Berendsen. 2007)

Short-term to sub-chronic toxicity studies are available and TCPP can be considered to affect body and liver weight and liver histopathology at or above 52 mg/kg/day (LOAEL).

 

Reproductive Toxicity:

The EU-RAR discussed the above mentioned 2-generation reproductive toxicity study results as follows: “In the two generation reproductive toxicity study with TCPP, an increase in oestrus cycle length and a decrease in uterus weight were observed in all dosed females in F0 generation and in high dose females in F1. The mean number of oestrus cycles was also increased in high dose animals of both generations. Effects were also noted on ovarian weights in all high dose females and pituitary weights in high dose females in F0 and all dosed females in F1. It is noted that all organ weight changes occurred in the absence of any histopathological changes, and it is accepted that uterine weight can fluctuate during the oestrus cycle. Therefore, the effects observed may be due to normal variation in cycling females.” (EU-RAR, 2008).

The authors of the study conclude that based on the effects on uterus weight in the F0-female animals, the low dose is considered to be the minimum effect dose for maternal toxicity. Based on the decreased number of pups delivered and the lower pup weight in the mid-dose group of the F1-generation, the low-dose was considered to be the no adverse effect level (NOAEL) for reproduction and developmental toxicity. The decrease of the uterus weights in females of the low and mid dose groups of the F0 generation was not accompanied by any microscopic findings in the uterus and no effect on fertility was seen in the low and mid dose. The effect may thus be judged as maternal toxicity but not as adverse with regard to fertility. In the high dose group the decrease in uterus weight was accompanied by a slightly abnormal cycle length, but no treatment related microscopical findings were observed (Waalkens-Berendsen. 2007).

 

Overall Conclusion on In Vivo Fertility Data

In the justification document for the CoRAP selection two in vivo effects are mentioned; decreased uterus weight and also prolongation of the oestrus cycle. As mentioned in the EU-RAR (2008) “It is noted that all organ weight changes occurred in the absence of any histopathological changes, and it is accepted that uterine weight can fluctuate during the oestrus cycle. Therefore, the effects observed may be due to normal variation in cycling females.” This interpretation is in agreement with the conclusions drawn by the study director and the observation may thus be judged as maternal toxicity but not as adverse with regard to fertility. (CoRAP Justification, 2015).

 

 

References:

 

Danish EPA. 2014. Larsen PB et al., Tris(2-chloro-1-methylethyl)phosphate; Survey, part of the Danish EPA Lous reviews

 

CoRAP justification. 2015. Justification for the selection of a substance for CoRAP inclusion. http://echa.europa.eu/documents/10162/93104ed5-f008-4d1d-8d8c-96a7056a59c0

 

EU-RAR. 2008. EU-Risk Assessment Report - Tris(2-chloro-1-methylethyl)phosphate (TCPP), CAS No. 13674-84-5)

 

Föllmann and Wober. 2006. Investigation of cytotoxic, genotoxic, mutagenic, and estrogenic effects of the flame retardants tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) in vitro. Toxicology Letters, 161(2), 124-134

 

Liu et al. 2012. Endocrine disruption potentials of organophophate flame retardants and related mechanisms in H295R and MVLN cell lined and in zebrafish. Aquatic Toxicology 114-115, 173-181, 2012

 

Kojima et al. 2013. In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors. Toxicology 314, 76-83, 2013

 

Leser. 1993. TRIS-CHLORISOPROPYLPHOSPHAT Subakute toxicologische Untersuchungen an Wistar Ratten (Verabreichung mit der Magensonde über 28 Tage). Internal Report No. 20213. Bayer AG

 

NTP Nonneoplastic Lesion Atlas, 2015. http://ntp.niehs.nih.gov/nnl/endocrine/thyroid/follhypert/index.htm

(accessed 13.10.2015)

 

NTP 2015. Testing information for Tris(chlorophropyl(phosphate – M20263. CAS-No. 13674-84-5. http://ntp.niehs.nih.gov/testing/status/agents/ts-m20263.html

Svenson et al. 2011. Antiestogenicity and estrogenicity in leachates from solid waste deposits. Environmental Toxicology 26, 233-239, 2011

 

Stauffer Chemical Company. 1981. Fyrol PCF 3-month dietary sub-chronic toxicity study in rats. Environmental Health Center.

 

Suzuki et al. 2013. Similarities in the endocrine-disrupting potencies of indoor dust and flame retardants by using human osteosarcoma (U2OS) cell based reporter gene assays. Environmental Science Technology 47, 1898-2908, 2013

 

US ATSDR. 2012. Toxicological Profile for Phosphate Ester Flame Retardants. Agency for Toxic Substances and Disease Registry, US Dep. of Health and Human Services, Atlanta Georgia, September 2012;

 

Waalkens-Berendsen. 2007. Oral two-generation reproduction toxicity study (including a dose range finding study) with Tris(2-chloro-1-methylethyl)-phosphate in rats. TNO Quality of Life, Report No. 031.32004

 

Zhang et al. 2014. Potential Estrogenic Effects of Phosphorus-Containing Flame Retardants. Environmental Science & Technology 48, 6995-7001, 2014