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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP, not conducted according to recognised guideline. Study is reported as literature report.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups (n = 12) of female rats, randomly selected based on body weight, received single daily oral doses of equal volume per kilogram body weight of either sesame oil, 0.4 g/kg TCP in sesame oil, or 1.7 g/kg neat BTP hydraulic fluid for 40 days. Rats were weighed weekly and doses adjusted accordingly for the following week. All rats were fasted 24 hr prior to sacrifice. All rats were sacrificed using halothane overdose and terminal body weights recorded. Adrenal glands and ovaries were removed, trimmed of all peripheral fat and fibrous connective tissue, weighed, and immediately placed in ice-cold homogenizing medium or formalin prior to microscopic and biochemical studies.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(methylphenyl) phosphate
EC Number:
215-548-8
EC Name:
Tris(methylphenyl) phosphate
Cas Number:
1330-78-5
Molecular formula:
C21H21O4P
IUPAC Name:
tris(4-methylphenyl) phosphate
Constituent 2
Reference substance name:
Tricresyl phosphate (TCP)
IUPAC Name:
Tricresyl phosphate (TCP)
Details on test material:
- Name of test material (as cited in study report): tricresyl phosphate
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: not specified
- Physical state: fluid
- Analytical purity: Compositions were consistent with typical production and free of any contaminants foreign to the chemistry of the materials.
- Impurities (identity and concentrations): see above
- Composition of test material, percentage of components: TCP was composed of mostly p and m-isomers of TCP and a mixture containing
substantial amounts of cresyl-xylyl, cresyl-ethylphenyl, and ethylphenylxylyl phosphates.
- Isomers composition: TCP was composed of mostly p and m-isomers of TCP
- Purity test date: not specified
- Lot/batch No.: not specified
- Expiration date of the lot/batch: not specified
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: not specified
- Storage condition of test material: not specified
- Other:

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals or test system and environmental conditions:
Designated virus-free, 10- 12 week-old female Fischer 344 rats weighing 100-160 g were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and maintained on rodent chow (Purina, St. Louis, MO) and tap water ad libitum for 12 days before experimentation. Rats were housed in our central animal holding facility in plastic cages, one rat per cage over untreated hardwood chip bedding. Temperature was maintained at 32 +/- 3°C and relative humidity at 50-70%. Lighting was controlled automatically to maintain a 12-hr light/dark cycle.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
other: sesame oil
Details on exposure:
Groups (n = 12) of female rats, randomly selected based on body weight, received single daily oral doses of equal volume per kilogram body weight of either sesame oil, 0.4 g/kg TCP in sesame oil, or 1.7 g/kg neat BTP hydraulic fluid for 40 days. Rats were weighed weekly and doses adjusted accordingly for the following week.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
40 days
Frequency of treatment:
single daily dose
Post exposure period:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0.4 g/kg TCP in sesame oil
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
All rats were fasted 24 hr prior to sacrifice. All rats were sacrificed using halothane overdose and terminal body weights recorded. Adrenal glands and ovaries were removed, trimmed of all peripheral fat and fibrous connective tissue, weighed, and immediately placed in ice-cold homogenizing medium or formalin.

Examinations

Examinations:
Adrenal glands and ovaries from two randomly chosen rats from each group were fixed in 10% buffered formalin, sectioned, embedded in paraffin, cut at 3-4 µm, stained with hematoxylin and eosin, and examined microscopically.

Adrenal and ovarian free cholesterol and CEs were extracted and separated by silicic acid-chromosorb W (acid washed)
column chromatography as previously described (Brown et al., 1975) except the lower phase was washed two additional times with pure upperphase solvent and CEs and cholesterol were eluted with 6 ml each of benzene and ethyl acetate, respectively; 15,000 dpm [3H]cholesteryl oleate and [14C]cholesterolr,e spectively, were added before extraction to monitor recovery and correct for procedural losses. Cholesterol esters were hydrolyzed in 400 p10.625 N ethanolic potassium hydroxide for 30 min at 80°C. Free cholesterol and cholesterol derived from isolated CEs were quantified
by the micromethod of Glick el al. (1964).

Protein was determined using a modified Bradford method (Bradford, 1976). All standards and samples were run in triplicate. Microsomal protein was assayed using a microassay protocol (Pierce Chemical Co., instruction booklet, 1989) after a 1:40 dilution of resuspended microsomes in homogenizing medium. A 2004 I: 1 sample volume to dye reagent was
analyzed for absorbance at 595 nm using a microtiter plate reader and pgfml protein determined usingalbumin-derived standards(PierceChemical Co.). A microtiter plate standard protocol (Pierce Chemical Co., instruction booklet, 1989) using a sample volume to dye reagent ratio of 150 was used to quantify cytosolic protein. A 204-p1 sample-reagent mixture
was read for absorbance at 595 nm and pglml protein determined using an albumin-derived standard curve.

Statistics. The results were analyzed by either one- or two-factorial analysis of variance (ANOVA). The analysis used the general linear models procedures ofSAS (SAS Institute, Cary, NC). When the Fvalue was significant, Scheffe's multiple comparison tests were used to determine group differences. Level of significance was set at either p < 0.05 or p < 0.0 1.
Positive control:
None

Results and discussion

Details on results:
Adrenal gland weights were significantly increased in the BTP and TCP-treated rats compared to controls and weights of the adrenal glands of the TCP group were statistically greater than those of the BTP group. There was an upward trend in the ovarian weight of BTP-treated rats, but only the TCP-treated rats had significantly increased ovarian weights compared to controls. Terminal body weights were not significantly different among any of the groups.
Microscopically the AC and OI cells from BTP- and TCP-treated rats had the consistently induced lesions previously characterized as cholestryl lipidosis (Latendresse et al., 1993a). The AC and OI cells were uniformly hypertrophied and the cytoplasmic area was filled with fine vacuoles. Adrenal glands and ovaries from the TCP group were more severely affected than those in the BTP-treated rats; however, both treated groups were different from controls.
Adrenal nCEH activity was significantly inhibited (p< 0.01) in rats receiving TCP (4% of controls) and BTP (44% of controls). Ovarian nCEH activity was decreased only in the TCP group. Activation by pretreatment of the nCEH with magnesium acetate, CAMP, ATP, and protein kinase A did not have a significant effect on nCEH activity (picomoles cholesterol formed per minute per milligram protein) within any of the groups.
ACAT activity in the adrenal glands was decreased to 73% of that of controls only in the TCP group with cholesterol. CE was significantly elevated in adrenal glands and ovaries from rats administered both TCP and BTP, with TCP having the most striking increase. Cholesterol was statistically increased only in the adrenal gland and ovaries from the TCP group.
The results of this study reveal a strong correlation between increased organ weights, severity of cholesteryl lipidosis, accumulation of CE, and the inhibition of nCEH activity in rats administered both TCP and BTP by comparing the effects of these two chemically related fluids at a single dose level and exposure time. TCP-exposed rats were the most severely affected for all end points. TCP-treated adrenal glands and ovaries had the greatest percentage of nCEH inhibition and the most severe cellular hypertrophy and lipidosis of AC and 01 cells. Previous time-course studies where rats were repeatedly dosed with 1.7 or 2.8 g/kg BTP and 0.4 g/kg TCP and sacrificed at 3, 6, and 9 weeks revealed that the severity of the lipidosis was progressive with histochemical and ultrastructural characteristics with both compounds that were consistent with an accumulation of CE (Latendresse et al., 1993a). TCP caused the most severe cholesteryl lipidosis in both AC and OI cells at all exposure intervals.
Ovarian homogenates from rats administered BTP did not have a decrease in nCEH activity in contrast to the adrenal gland, but they did have a significant increase in CE concentration. Affected OI cells contributed a smaller proportion of the total tissue volume in the ovary compared to the AC cells of the adrenal gland. Because ACAT and nCEH assays were performed using whole tissue homogenates, a potential inhibition of nCEH in ovaries of BTP treated rats was not detected, most likely due to the relatively small proportion of interstitial cells in the ovary and the milder effect of BTP compared to TCP. The elevated CE concentration in the ovaries with cholesteryl lipidosis of OI cells as measured by a sensitive microanalysis method supports this interpretation along with the significant inhibition of nCEH in the adrenal glands of the same rats. The CE concentration increased significantly in the adrenal glands and ovaries in TCP- and BTP treated rats with nCEH inhibition.
Triaryl phosphates have been reported to inhibit several esterases in rats, including nonspecific tissue esterase (Somkuti et al., l987), neurotoxic esterase (Somkuti et al., 1987), and cholinesterase (TCP and/or BTP) (Hejtmancik et al., 1986; Kinkead et al., 1993) in rats in a dose-response manner. In the present study, nCEH activity was determined at a single dose level and time-point for these chemically related compounds. It cannot be interpreted with certainty that the cholesteryl Iipidosis induced by BTP and TCP in the adrenal gIand and ovary was only caused by nCEH inhibition.
However, the correlation between the degree of nCEH inhibition and the severity of the cholesteryl lipidiosis detected by comparing BTP and TCP at a single dose and time, coupled with identical histologic, histochemical, and biochemical lesions in the same target tissues for both compounds, and the known inhibitory effects of triaryl phosphates on other esterases strongly suggests an inhibition of nCEH as the most likely cause of the cholesteryl lipidosis for both compounds.
The decrease in ACAT activity (27%) in the adrenal glands of TCP-treated rats compared to that of controls was associated with an increase ( 4.3X ) in CE. The suppressed ACAT activity also was accompanied by a significant increase in cholesterol in adrenal glands of rats administered
TCP. Cholesterol was elevated in the ovaries of the same rats without a detectable suppression of ACAT activity, most likely due to the relative proportion of affected cells compared to many nonaffected cells in the ovaries. Cholesterol levels were altered in the direction expected based on lower ACAT activity. Suppression of ACAT activity over time would account for an elevation in free cholesterol. A decrease in ACAT specific activity may not be the sole mechanism for the increased cholesterol in AC and OI cells. Factors that regulate ACAT activity include tropic hormone-induced downregulation, availability of intracellular substrate, and functioning of the LP-uptake pathway(s) (Suckling and Stange, 1985; Pedersen, 1988). The mechanism by which ACAT activity is suppressed by TCP in adrenal glands is unknown at present.

Applicant's summary and conclusion

Conclusions:
In conclusion, triaryl phosphates caused inhibition of nCEH in the adrenal gland and ovary in rats. The nCEH inhibition most likely resulted in a defect in the cholesterol storage pathway contributing to accumulation of CE in cytoplasmic lipid droplets, and may have inhibited conversion of CE from HDL to free cholesterol. This important enzyme mobilizes cholesterol in CE to form free cholesterol needed for steroidogenesis in AC and OI cells. To our knowledge, triaryl phosphates are the first chemicals proposed to cause cholesteryl lipidosis as a result of a defect in the cholesterol storage pathway without inhibition of steroidogenesis in rats. Further studies on triaryl phosphates are needed to determine a dose producing no effect in rats, identify potential clinical biomarkers of exposure in human patients, investigate reversibility of the lesion, determine the toxicity by dermal exposure (most significant route of exposure of personnel in the workplace), and to evaluate adrenocortical function under stressful conditions.
Executive summary:

The objectives of this study were to determine if the administration of triaryl phosphate fluids caused a defect in the cholesterol

storage pathway of AC and OI cells and to determine the mechanism of action. Female rats received daily oral doses of 0 or 0.4

glkg TCP in sesame oil vehicle or 1.7 glkg neat BTP for 40 days. Adrenal glands from both treatment groups and ovaries from

TCP-treated rats were heavier than controls. Microscopic and biochemical studies revealed cholesteryl lipidosis composed of

CE in the adrenal glands and ovaries in BTP- and TCP-treated rats with the latter group affected most severely. The activity of

neutral CE hydrolase (nCEH), an enzyme that converts CE to cholesterol in the uptake and storage pathways, also was inhibited

most in the TCP-treated group (97% inhibition compared to that of control). The activity of acyl coenzyme A:cholesterol

acyl transferase, an enzyme that esterifies cholesterol to make CE, was depressed 27% compared to that of control adrenal

glands of the TCP group, resulting in elevated intracellular cholesterol levels in AC cells. An inhibition of nCEH in the storage

and uptake pathways by triaryl phosphates most likely resulted in the striking accumulation of CE in cytoplasmic lipid droplets

of AC and 01 cells in F344 rats.

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