Tris(chloropropyl) phosphate (TCPP) is used as a flame retardant in textiles, furniture foam, and other related products. In addition, it is manufactured for use in construction materials, electronic products, paints, coatings, and adhesives. Several flame retardants, including structurally similar organohalogen compounds, have been removed from products in commerce due to toxicity concerns, and TCPP has been proposed as a replacement flame retardant for use in these products. An anticipated increase in use of TCPP has generated concerns for increased human exposure through oral, dermal, and inhalation routes; however, publicly available toxicity data are scarce. The U.S. Consumer Product Safety Commission therefore requested that the National Toxicology Program (NTP) form a research program on TCPP to conduct subchronic and chronic exposure studies in rats and mice for hazard identification and characterization information. Because TCPP is commercially available as an isomeric mixture, the NTP studies tested a commercial TCPP product containing four isomers commonly found in other commercial mixtures of TCPP: tris(1-chloro-2-propyl) phosphate (TCIPP; CASRN 13674-84-5), bis(2-chloro-1-methylethyl) 2-chloropropyl phosphate (CASRN 76025-08-6), bis(2-chloropropyl) 2-chloroisopropyl phosphate (CASRN 76649-15-5), and tris(2-chloropropyl) phosphate (CASRN 6145-73-9). Following procurement of TCPP, the percent purity of the four isomers was determined prior to conducting hazard characterization studies.
In the subchronic toxicity studies of TCPP in male and female Sprague Dawley (Hsd:Sprague Dawley SD) rats and B6C3F1/N mice, animals were exposed via dosed feed for 3 months. In rats, perinatal TCPP exposure of time-mated females from gestation day (GD) 6 through postnatal day (PND) 21 (weaning) preceded the subchronic exposure. Exposure concentrations for these studies were selected based on palatability studies conducted as part of the NTP research program on TCPP and on industry reports. Pregnant rats (20 dams) were exposed to 0, 2,500, 5,000 (8 dams only), 10,000, or 20,000 (8 dams only) ppm TCPP throughout gestation and lactation. Groups of 10 rats/sex/exposure concentration continued on study after weaning and were fed diets containing the same respective TCPP concentrations as their respective dam for 3 months. Mice (10/sex/exposure concentration) were exposed to 0, 1,250, 2,500, 5,000, 10,000, or 20,000 ppm TCPP for 3 months. Toxicity was evaluated by assessing survival, clinical observations, body weight, and feed consumption in all rats (including during the perinatal exposure period) and mice for 3 months. At study termination, additional toxicity parameters—including organ weight, hematology and clinical chemistry (rats only), sperm motility (males), genetic toxicity, and histopathology—were evaluated in rats and mice. The results of the 3-month studies were used to design and select exposure concentrations for the 2-year studies in rats and mice.
For the chronic toxicity studies, time-mated female rats were provided dosed feed beginning on GD 6 through lactation. On PND 28, offspring (50/sex/group) continued on the study and were provided dosed feed containing the same TCPP concentration as their respective dam for 2 years. In mice, groups of 50 mice per sex, aged 5 to 6 weeks at study start, were provided dosed feed containing TCPP for 2 years. At study termination, toxicity (e.g., survival, body weights) and the incidence of neoplasms and chemical-related histopathological changes were evaluated in rats and mice.
Three-month study in rats
In the perinatal portion of the 3-month study, pregnant rats exposed to 40,000 ppm were humanely euthanized due to overt toxicity early in gestation. With the exception of sporadic decreases in maternal body weight and feed consumption during gestation and lactation (approximately 10%–20% lower than control rats), no other toxicologically relevant findings were reported for dams. TCPP exposure also had no effects on littering parameters at concentrations ≤20,000 ppm, and offspring survived through lactation. Offspring in the 20,000 ppm TCPP group did exhibit a time-dependent decrease in weight gain during lactation. Male offspring in the 20,000 ppm group failed to thrive after weaning and were removed from the subchronic portion of the study on day 5; females in this exposure group were kept on study.
For the remainder of the 3-month study, male and female rats survived and displayed no clinical signs of toxicity when exposed to 10,000 ppm TCPP or lower concentrations. Females in the 20,000 ppm TCPP group had a mean body weight that was 12% lower than that of control females, which corresponded with a similar decrease in feed consumption (18%) by study termination. No biologically relevant alterations in hematological parameters were observed in either sex. Serum cholesterol concentrations were significantly increased in both sexes. TCPP did elicit exposure concentration-related effects (i.e., significantly increased organ weights and microscopic changes) in the liver and thymus of rats. In the liver, bile duct hyperplasia was observed in the highest exposure groups for both sexes. Increases in thymus weight were correlated with significantly larger thymic cortices in all males exposed to TCPP and in females in the 10,000 ppm group.
Two-year study in rats
The effect of chronic TCPP exposure was evaluated in rats, beginning in utero and through adulthood, following feed administration at target concentrations of 0, 2,500, 5,000, 10,000, or 20,000 ppm TCPP. TCPP exposure to dams had no toxicologically relevant effects on maternal measurements during gestation or lactation with the exception of a slightly lower mean body weight and feed consumption in the 20,000 ppm group over this perinatal period. An exposure concentration-related decrease in mean body weight relative to control animals was observed in male and female offspring in the 20,000 ppm TCPP group during lactation. At the end of the 2 year study, mean body weights of males and females in the 20,000 ppm group were 8% and 17% lower, respectively, than those of the control groups. Histopathological evaluations identified a positive trend for incidences of hepatocellular adenoma or carcinoma (combined) in male rats. Accompanying significant nonneoplastic lesions included hyperplasia of the bile duct and an increase in basophilic, eosinophilic, mixed-cell foci, and pigment in the liver of males exposed to 20,000 ppm TCPP. A nonsignificant increase in the incidence of hepatocellular adenomas was observed in females exposed to 2,500, 10,000, and 20,000 ppm TCPP, and a spectrum of nonneoplastic lesions, similar to those in male rats, was observed. Histopathological evaluations also identified a positive trend for incidences of uterine adenoma or adenocarcinoma (combined) in female rats, although this was not significant at any exposure concentration.
Three-month study in mice
In the 3-month TCPP study with mice, mortality and clinical signs of toxicity were not observed across the exposure groups ranging from 1,250 to 20,000 ppm. TCPP-exposed male and female mice gained less weight than control mice; however, this response was only concentration related in the males. At the end of the subchronic study, TCPP exposure was associated with a spectrum of organ weight changes and microscopic changes in both sexes. Significantly increased liver weights were observed alongside a significant increase in the incidences of hepatocellular hypertrophy in males and females exposed to 5,000, 10,000, and 20,000 ppm TCPP. A significant increase in the incidences of cytoplasmic alteration was observed in the renal tubules of male mice exposed to 2,500, 5,000, 10,000, and 20,000 ppm TCPP. This observation was not evident in females and was not correlated with an observed decrease in kidney weights of both sexes.
Two-year study in mice
The TCPP exposure groups were different for male and female mice exposed chronically. Exposure concentrations of TCPP in feed were 0, 1,250 (males only), 2,500, 5,000, or 10,000 (females only) ppm. An exposure concentration-related decrease in mean body weights was recorded in males and females relative to their respective control groups; however, survival, clinical observations, and feed consumption measurements were not suggestive of overt toxicity. Lower mean body weight was interpreted as a failure to gain weight. Similar to rats, mice also had a significant increase in liver neoplasms. Male mice had a significant increase in the incidence of hepatocellular carcinoma across all TCPP-exposed groups, but the incidences were similar among these groups. Significant increases in the incidence of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) were also noted in the 10,000 ppm TCPP-exposed female mice relative to the control females. Exposure-related nonneoplastic lesions were not observed in male mice. However, a significant increase in cytoplasmic alteration of hepatocytes was observed in nearly all females of the 10,000 ppm group. Additionally, a significant increase in eosinophilic foci was recorded in all female TCPP-exposed groups.
Genetic toxicology
In two independent studies, TCPP was not mutagenic in any of several strains of bacteria in tests conducted with and without rat or hamster liver S9 fraction. In the in vivo rodent peripheral blood micronucleus assay, no increases in micronucleated erythrocytes were observed in male or female Sprague Dawley rats administered TCPP via dosed feed. Results of the in vivo micronucleus assay in B6C3F1/N female mice were also judged to be negative. In male mice, a small but significant increase in micronucleated mature erythrocytes, accompanied by a small increase in the micronucleated immature erythrocyte population, resulted in an equivocal call. In both rats and mice, the percentage of immature erythrocytes increased in a dose-related manner, suggesting a stimulation of erythropoiesis.
Conclusions
Under the conditions of these 2-year feed studies, there was some evidence of carcinogenic activity of tris(chloropropyl) phosphate (TCPP) in male Hsd:Sprague Dawley SD rats based on the increased incidence of hepatocellular adenoma or carcinoma (combined). There was some evidence of carcinogenic activity of TCPP in female Hsd:Sprague Dawley SD rats based on the increased incidence of uterine adenoma or adenocarcinoma (combined). The marginal increase in the incidence of hepatocellular adenoma in female rats may have been related to exposure.
There was some evidence of carcinogenic activity of TCPP in male B6C3F1/N mice based on the increased incidence of hepatocellular carcinoma. There was clear evidence of carcinogenic activity of TCPP in female B6C3F1/N mice based on the increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined).
In the 2-year studies, exposure to TCPP resulted in increased incidences of nonneoplastic lesions in the liver of male and female rats and in female mice, and in the kidney of male mice.
National Toxicology Program (NTP). 2023. NTP technical report on the toxicology and carcinogenesis studies of an isomeric mixture of tris(chloropropyl) phosphate administered in feed to Sprague Dawley (Hsd:Sprague Dawley SD) rats and B6C3F1/N mice. Research Triangle Park, NC: National Toxicology Program. Technical Report 602. https://doi.org/10.22427/NTP-TR-602