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Endpoint:
specific investigations: other studies
Remarks:
Uterotrophic Assay
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1600
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD, Section 4 (Part 440): Uterotrophic Bioassay in Rodents, Guideline for the Testing of Chemicals
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other:
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina
- Age at study initiation: ~ 65 days old
- Weight at study initiation: 247.4 to 306.9 g
- Fasting period before study: No data
- Housing: Animals were housed 2 – 3 per cage in solid bottom caging with Shepherd’s™ ALPHA-dri® bedding (Animal Specialties and Provisions LLC, Quakertown, Pennsylvania, U.S.A., catalog number SHE1009), a loose bedding comprised of pure alpha cellulose. Nestlets were provided as enrichment.
- Diet (e.g. ad libitum): Harlan Teklad 2016 certified feed (Madison, Wisconsin, U.S.A.)
- Water (e.g. ad libitum): Tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
other: 0.1% Tween® 80 in 0.5% methylcellulose
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 days
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
Five groups of young adult female ovariectomized Crl:CD(SD) rats (15/group) were dosed by oral gavage with a suspension of 0, 300, or 500 mg/kg/day of the test substance administered in the vehicle (0.1% Tween® 80 in 0.5% methylcellulose), for 6 consecutive days, and sacrificed approximately 2-4 hours after the last administered dose. Rats in the negative control group received vehicle only (0.1% Tween® 80 in 0.5% methylcellulose). Two separate ovariectomized positive control groups, one group administered 0.1 mg/kg/day of the estrogen receptor agonist 17α-ethynyl estradiol dissolved in vehicle (corn oil with 1% ethanol) and one administered 20 mg/kg/day of the dopamine (D2) receptor agonist bromocriptine mesylate suspended in vehicle (0.1% Tween® 80 in 0.5% methylcellulose) were included to verify test system performance.
Examinations:
Vaginal cytology was evaluated daily to assess the potential of the test substance to induce cytological changes consistent with those observed with the 17α-ethynyl estradiol positive control. At scheduled euthanasia, uterine weights were collected in order to assess the ability of the test substance to induce uterine growth. Blood was collected at the time of sacrifice from all animals for serum prolactin concentration analysis.
Positive control:
Yes, 17α-ethynyl estradiol dissolved in vehicle (corn oil with 1% ethanol) and bromocriptine mesylate suspended in vehicle (0.1% Tween® 80 in 0.5% methylcellulose)
Details on results:
The test substance did not induce changes on any parameters consistent with the potential to act as an estrogen receptor agonist (i.e., increased uterine weight, uterine fluid imbibition, estrous cycle conversion from diestrous to estrous) in ovariectomized adult female rats. However, the test substance reduced serum prolactin concentrations, similar to the effect observed with the D2 receptor agonist (positive control chemical) bromocriptine mesylate. Under the conditions of the study, the test substance did not act as an estrogen receptor agonist, but reduced serum prolactin concentrations in ovariectomized adult female rats when administered 300 and 500 mg/kg/day for 6 consecutive days.
Conclusions:
The test substance did not induce changes on any parameters consistent with the potential to act as an estrogen receptor agonist (i.e., increased uterine weight, uterine fluid imbibition, estrous cycle conversion from diestrous to estrous) in ovariectomized adult female rats. Under the conditions of the study, the test substance did not act as an estrogen receptor agonist, but reduced serum prolactin concentrations in ovariectomized adult female rats when administered 300 and 500 mg/kg/day for 6 consecutive days.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to act as an estrogen receptor agonist or prolactin modulator when administered by oral gavage to ovariectomized rats for 6 consecutive days (U.S. EPA Health Effects Test Guidelines OPPTS 890.1600 and OECD, Section 4 (Part 440): Uterotrophic Bioassay in Rodents, Guideline for the Testing of Chemicals).

 

Five groups of young adult female ovariectomized Crl:CD(SD) rats (15/group) were dosed by oral gavage with a suspension of 0, 300, or 500 mg/kg/day of the test substance administered in the vehicle (0.1% Tween® 80 in 0.5% methylcellulose), for 6 consecutive days, and sacrificed approximately 2-4 hours after the last administered dose. Rats in the negative control group received vehicle only (0.1% Tween® 80 in 0.5% methylcellulose). Two separate ovariectomized positive control groups, one group administered 0.1 mg/kg/day of the estrogen receptor agonist 17α-ethynyl estradiol dissolved in vehicle (corn oil with 1% ethanol) and one administered 20 mg/kg/day of the dopamine (D2) receptor agonist bromocriptine mesylate suspended in vehicle (0.1% Tween® 80 in 0.5% methylcellulose) were included to verify test system performance. Samples of each test substance dose preparation were collected near the beginning of the study and analyzed to verify concentration; test substance stability had been previously established. All test substance dose groups were at the acceptable concentrations. The dose formulations for the positive control chemicals were not collected or evaluated. Body weights and clinical observations were recorded daily. Food consumption was recorded on test day 1 and test day 6. Vaginal cytology was evaluated daily to assess the potential of the test substance to induce cytological changes consistent with those observed with the 17α-ethynyl estradiol positive control. At scheduled euthanasia, uterine weights were collected in order to assess the ability of the test substance to induce uterine growth. Blood was collected at the time of sacrifice from all animals for serum prolactin concentration analysis.

 

No test substance-related mortality occurred in this study. There were test substance-related clinical observations of stained bedding the color of the test article in all rats in the 300 mg/kg/day and 500 mg/kg/day group on test days 2-6 and 1-6, respectively. Mean body weight gain (test days 1 to 6) was statistically significantly decreased in rats administered 300 and 500 mg/kg/day, and mean final body weights were statistically significantly decreased by 7% and 11% in the 300 mg/kg/day and 500 mg/kg/day, respectively, when compared to the negative control group mean. The effects on body weight in the 300 mg/kg/day and 500 mg/kg/day dose groups were accompanied by decreases in mean daily food consumption and mean daily food efficiency. Mean daily food consumption was 34% lower and 54% lower in the 300 mg/kg/day and 500 mg/kg/day, respectively, when compared to the negative control group. Mean daily food efficiency was 136% and 299% lower when compared to the negative control group, respectively. All animals receiving the test substance remained in diestrus for the duration of the study. At scheduled euthanasia, there were no gross observations noted nor were there any treatment-related effects on uterine weight at any level tested. Serum prolactin levels were decreased in rats administered 300 and 500 mg/kg/day (32% and 60% decrease compared to the negative control group for the 300 and 500 mg/kg/day dose groups, respectively), although only the decrease at 500 mg/kg/day was statistically significant.

 

In rats administered the positive control chemical, 17α-ethynyl estradiol, no mortality occurred and there were no clinical observations noted during in this study. Mean body weight gain and mean final body weights were decreased as compared to concurrent negative control group. The body weight effects were accompanied by decreased food consumption and food efficiency. All 15 rats administered 17α-ethynyl estradiol showed effects on the stage of estrous. On test day 4, all 15 rats administered 17α-ethynyl estradiol showed cytological markers indicative of either proestrus or estrus. At scheduled euthanasia, 12 out of 15 rats showed the presence of uterine fluid within the uterus. Absolute uterine wet weight and blotted weight were increased to 198% and 162% of the negative control, respectively. Relative (to final body weight) uterine wet weight and blotted weight were increased to 226% and 186% of the negative control, respectively. Serum prolactin levels were increased 82% as compared to the negative control values. As expected, rats administered 17α-ethynyl estradiol showed effects consistent with an estrogen receptor agonist.

 

In rats administered the positive control chemical, bromocriptine mesylate, no mortality occurred in this study. The clinical observation of eyelid ptosis was observed in all rats at approximately 2 hours post-dose on test days 1-5. Mean body weight gain and mean final body weights were decreased as compared to the negative control values. The body weight effects were accompanied by decreased food consumption and food efficiency. All animals receiving the bromocriptine mesylate remained in diestrus for the duration of the study. At scheduled euthanasia, there were no gross observations noted nor were there any treatment-related effects on uterine weight. Serum prolactin levels were decreased by 98% as compared to the negative control values. The results with bromocriptine mesylate are consistent with a D2 receptor agonist.

 

In conclusion, the test substance did not induce changes on any parameters consistent with the potential to act as an estrogen receptor agonist (i.e., increased uterine weight, uterine fluid imbibition, estrous cycle conversion from diestrous to estrous) in ovariectomized adult female rats. However, the test substance reduced serum prolactin concentrations, similar to the effect observed with the D2 receptor agonist (positive control chemical) bromocriptine mesylate. Under the conditions of the study, the test substance did not act as an estrogen receptor agonist, but reduced serum prolactin concentrations in ovariectomized adult female rats when administered 300 and 500 mg/kg/day for 6 consecutive days.

Endpoint:
endocrine system modulation
Remarks:
Radioligand binding
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Radioligand binding is widely used to characterize receptors and determine their anatomical distribution. The affinity and selectivity of an unlabeled ligand to compete for the binding of a fixed concentration of a radiolabeled ligand to a receptor are determined using a competition binding assay. Methods employed in this study have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained.

IC50 values were determined by a non-linear, least squares regression analysis using MathIQTM (ID Business Solutions Ltd., UK). Where inhibition constants (Ki) are presented, the Ki values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol., 22:3099-3108, 1973) using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the Ko of the ligand (obtained experimentally at Eurofins Panlabs, Inc.). Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using MathlQ™. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other:
Species:
other: In vitro
Route of administration:
other: in vitro
Analytical verification of doses or concentrations:
no
Details on results:
IC50 values were > 10 µM in all the assays. The test substance has no activity.
Conclusions:
IC50 values were > 10 µM in all the assays. The test substance has no activity.
Executive summary:

Radioligand binding is widely used to characterize receptors and determine their anatomical distribution. The affinity and selectivity of an unlabeled ligand to compete for the binding of a fixed concentration of a radiolabeled ligand to a receptor are determined using a competition binding assay.

Significant results are displayed in the following table(s) in rank order of potency for estimated IC50 and/or Ki values. For primary assays, only the lowest concentration with a significant response judged by the assays' criteria, is shown in this summary.

 

Receptor                                           IC50

 

Dopamine D1                                    > 10.0µM

Dopamine D2L                                   > 10.0µM

Dopamine D2S                                    > 10.0µM

Dopamine D3                                      > 10.0µM

Dopamine D4.2                                    > 10.0µM

Dopamine D4.4                                    > 10.0µM

Dopamine D4.7                                    > 10.0µM

Dopamine D5                                      > 10.0µM

Melatonin MT, Non-Selective             > 10.0µM

Melatonin MT1                                    > 10.0µM

Melatonin MT2                                    > 10.0µM

Transporter, Dopamine (DAT)           > 10.0µM

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to evaluate the ability of the test substance to bind to testosterone or estrogen receptors. Competitive binding assays measure the binding of the radioligand to the receptors with increasing concentrations of a test substance. The concentration at which the test substance displaces half of the bound radioligand is the IC50 (often expressed as logIC50).

An experimental run was performed to evaluate the test substance for its ability to compete with [³H]-Methyltrienolone in binding to the human testosterone receptor in LnCAP cellsab and with 17-β-Estradiol in binding to the human estrogen receptor in MCF-7 cellscd in vitro.

The test substance was evaluated at 8 concentrations between 1.0 x 10-8 and 1.0 x 10-4 M. Radioinert Methyltrienolone, the testosterone receptor agonist reference standard, and radioinert 17-β-Estradiol, the estrogen receptor agonist reference standard were used as the positive control to verify test system performance.

A test substance is considered an “active” binder if it exhibits inhibition of greater than 50% at any concentration and is typically accompanied by a dose-dependent response. A test substance is considered a “marginal” binder if it exhibits inhibition between 20 and 49%. A test substance is considered “inactive” if it ranges between -20% to + 20 % inhibition.
GLP compliance:
no
Remarks:
The work described in this report is not required to be in compliance with U.S. EPA FIFRA (40 CFR part 160) Good Laboratory Practice Standards, which are compatible with current OECD and MAFF (Japan) Good Laboratory Practices.
Type of method:
in vitro
Endpoint addressed:
other: Ability to bind to testosterone or estrogen receptors
Species:
other: In vitro
Route of administration:
other: In vitro
Analytical verification of doses or concentrations:
no
Details on results:
The test substance did not competitively bind to the testosterone or estrogen receptors when tested up to a maximum concentration of 1.0 x 10-4 M. Therefore, under the conditions of the study, the test substance is classified as a non-inhibitor in the testosterone and estrogen receptor binding assay.
Conclusions:
Under the conditions of the study, the test substance is classified as a non-inhibitor in the testosterone and estrogen receptor binding assay.
Executive summary:

The objective of this study was to evaluate the ability of the test substance to bind to testosterone or estrogen receptors. Competitive binding assays measure the binding of the radioligand to the receptors with increasing concentrations of a test substance. The concentration at which the test substance displaces half of the bound radioligand is the IC50 (often expressed as logIC50).

 

An experimental run was performed to evaluate the test substance for its ability to compete with [³H]-Methyltrienolone in binding to the human testosterone receptor in LnCAP cellsab and with 17-β-Estradiol in binding to the human estrogen receptor in MCF-7 cellscd in vitro.

 

The test substance was evaluated at 8 concentrations between 1.0 x 10-8 and 1.0 x 10-4 M. Radioinert Methyltrienolone, the testosterone receptor agonist reference standard, and radioinert 17-β-Estradiol, the estrogen receptor agonist reference standard were used as the positive control to verify test system performance.

 

As expected, radioinert Methyltrienolone showed effects consistent with strong competitive binding to the testosterone receptor. The logIC50 was determined to be approximately 1.22 x 10-9 M for radioinert methyltrienolone. Radioinert 17-β-Estradiol showed effects consistent with strong competitive binding to the estrogen receptor. The logIC50 was determined to be approximately 3.28 x 10-10M.

 

The test substance did not competitively bind to the testosterone or estrogen receptors when tested up to a maximum concentration of 1.0 x 10-4 M. Therefore, under the conditions of the study, the test substance is classified as a non-inhibitor in the testosterone and estrogen receptor binding assay.

Endpoint:
hepatotoxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to evaluate liver mechanistic parameters (microscopic pathology, cell proliferation, and induction of cytochrome P450 enzyme activities and gene expression) that are possibly associated with liver effects observed in feeding studies in mice exposed to the test substance.

Six groups of young adult male Crl:CD1(ICR) mice were administered diets incorporating 0, 200, 800, 2500 or 7000 ppm of the test substance, or 1000 ppm of a positive control substance, phenobarbital. Ten male mice per group were sacrificed at each time point on test days 3, 8, and 29 (equivalent to 2, 7, or 28 days of dietary exposure, respectively, due to initiation of exposure on test day 1).

The diets were analyzed to verify that the test substance was homogeneously mixed at the targeted concentrations for all dietary concentrations. Diets were used within the period of previously established stability.

Body weights, food consumption, and clinical observations were collected weekly and at sacrifice. Livers from all animals were evaluated for microscopic pathology (using hematoxylin and eosin staining), cell proliferation (Ki67 labeling), and induction of cytochrome P450 enzyme activities. Gene expression for cytochrome P450 enzymes was evaluated in livers from the control, 7000 ppm test substance, and positive control groups on day 3 and day 8.
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other: Hepatotoxicity
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc, Raleigh, North Carolina, USA.
- Age at study initiation: ~ 7 weeks old
- Weight at study initiation: Control: 30.2 g; 200 ppm: 30.3 g; 800 ppm: 30.5 g; 2500 ppm: 30.5 g; 7500 ppm: 29.6 g; Positive control: 30.1 g
- Fasting period before study: No
- Housing: Animals were housed individually in solid bottom caging with bedding and species-appropriate enrichment.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
oral: feed
Vehicle:
other: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples containing the test substance were submitted for homogeneity and the concentration verification analyses. Diet samples were diluted with acetonitrile before analysis by ultra-high-performance liquid chromatography with ultraviolet detection.
The ultra-high-performance liquid chromatography with ultraviolet detection instrument was configured as follows:

Instrument: Agilent 1290 Infinity liquid chromatograph
Software: OpenLAB CDS ChemStation Ed. Rev. C.01.05[35]
Column: Thermo ODS Hypersil, 5 µM, 100 x 2.1 mm
Mobile Phase: 60% A: water containing 3.1 mM phosphoric acid and 40% B: acetonitrile
Flow Rate: 0.4 mL/min
Stop Time: 5 minutes
Detection: UV absorbance at 234 nm
Colum Temperature: 30.0°C
Injection Volume: 1 µL
Duration of treatment / exposure:
2, 7, or 28 days
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
200 ppm
Dose / conc.:
800 ppm
Dose / conc.:
2 500 ppm
Dose / conc.:
7 000 ppm
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Examinations:
Body weights, food consumption, and clinical observations were collected weekly and at sacrifice. Livers from all animals were evaluated for microscopic pathology (using hematoxylin and eosin staining), cell proliferation (Ki67 labeling), and induction of cytochrome P450 enzyme activities. Gene expression for cytochrome P450 enzymes was evaluated in livers from the control, 7000 ppm test substance, and positive control groups on day 3 and day 8.
Positive control:
Yes, phenobarbital
Details on results:
All animals survived to scheduled sacrifice. The overall mean daily intake of the test substance at 200, 800, 2500, and 7000 ppm was calculated as 35, 149, 420, and 1210 mg/kg/day, respectively, for the day 3 subset; 35, 151, 450, and 1275 mg/kg/day, respectively, for the day 8 subset; and 32, 133, 411, and 1273 mg/kg/day, respectively, for the day 29 subset.

Under the conditions of this study, exposure of male mice to the test substance at a dietary concentration of 7000 ppm resulted in hepatocellular centrilobular hypertrophy, increased liver weights, and increased hepatocellular proliferation. These liver changes occurred in association with induction of cytochrome P450 enzyme activities and gene expression consistent with that seen with the prototypical constitutive androstane receptor (CAR) inducer phenobarbital, with the most pronounced changes occurring in CYP2B10 gene expression. These effects were usually observed at all time points evaluated (day 3, 8, and 29, where applicable), except for the increase in hepatocellular proliferation, which was only observed on day 8. Mice administered 2500 ppm did not exhibit any increase in hepatocellular proliferation, but did exhibit other liver effects (hepatocellular centrilobular hypertrophy, increased liver weights, and minimal induction of cytochrome P450 enzyme activities), with generally lower magnitude or lower incidence relative to the 7000 ppm group. There were no biologically relevant effects at 200 or 800 ppm with respect to any parameter or time point evaluated.

The changes observed in the cytochrome P450 profile, along with the associated changes in the livers of male mice at 7000 ppm, support a phenobarbital-like mechanism of action for liver tumor induction observed in male mice following chronic exposure to that dietary concentration. The first key event in this mechanism is induction of CYP2B enzymes with resulting liver hypertrophy and increased liver weights. These changes are followed by the second key event of liver cell proliferation progressing to liver tumors with chronic exposure. Consistent with this mechanism, the second key event of increased liver cell proliferation was only seen at the 7000 ppm concentration in the current study, which is the only concentration that produced liver tumors in the 18-month mouse feeding study.

The analysis results show the test substance was homogeneously mixed at the targeted concentrations for all diet samples analyzed. The test substance was not detected in the control diet.

Conclusions:
Under the conditions of this study, exposure of male mice to the test substance at a dietary concentration of 7000 ppm resulted in hepatocellular centrilobular hypertrophy, increased liver weights, and increased hepatocellular proliferation.
Executive summary:

The objective of this study was to evaluate liver mechanistic parameters (microscopic pathology, cell proliferation, and induction of cytochrome P450 enzyme activities and gene expression) that are possibly associated with liver effects observed in feeding studies in mice exposed to the test substance.

 

Six groups of young adult male Crl:CD1(ICR) mice were administered diets incorporating 0, 200, 800, 2500 or 7000 ppm of the test substance, or 1000 ppm of a positive control substance, phenobarbital. Ten male mice per group were sacrificed at each time point on test days 3, 8, and 29 (equivalent to 2, 7, or 28 days of dietary exposure, respectively, due to initiation of exposure on test day 1).

 

The diets were analyzed to verify that the test substance was homogeneously mixed at the targeted concentrations for all dietary concentrations. Diets were used within the period of previously established stability. The analysis results show the test substance was homogeneously mixed at the targeted concentrations for all diet samples analyzed. The test substance was not detected in the control diet.

 

Body weights, food consumption, and clinical observations were collected weekly and at sacrifice. Livers from all animals were evaluated for microscopic pathology (using hematoxylin and eosin staining), cell proliferation (Ki67 labeling), and induction of cytochrome P450 enzyme activities. Gene expression for cytochrome P450 enzymes was evaluated in livers from the control, 7000 ppm test substance, and positive control groups on day 3 and day 8.

 

All animals survived to scheduled sacrifice. The overall mean daily intake of the test substance at 200, 800, 2500, and 7000 ppm was calculated as 35, 149, 420, and 1210 mg/kg/day, respectively, for the day 3 subset; 35, 151, 450, and 1275 mg/kg/day, respectively, for the day 8 subset; and 32, 133, 411, and 1273 mg/kg/day, respectively, for the day 29 subset.

 

Under the conditions of this study, exposure of male mice to the test substance at a dietary concentration of 7000 ppm resulted in hepatocellular centrilobular hypertrophy, increased liver weights, and increased hepatocellular proliferation. These liver changes occurred in association with induction of cytochrome P450 enzyme activities and gene expression consistent with that seen with the prototypical constitutive androstane receptor (CAR) inducer phenobarbital, with the most pronounced changes occurring in CYP2B10 gene expression. These effects were usually observed at all time points evaluated (day 3, 8, and 29, where applicable), except for the increase in hepatocellular proliferation, which was only observed on day 8. Mice administered 2500 ppm did not exhibit any increase in hepatocellular proliferation, but did exhibit other liver effects (hepatocellular centrilobular hypertrophy, increased liver weights, and minimal induction of cytochrome P450 enzyme activities), with generally lower magnitude or lower incidence relative to the 7000 ppm group. There were no biologically relevant effects at 200 or 800 ppm with respect to any parameter or time point evaluated.

 

The changes observed in the cytochrome P450 profile, along with the associated changes in the livers of male mice at 7000 ppm, support a phenobarbital-like mechanism of action for liver tumor induction observed in male mice following chronic exposure to that dietary concentration. The first key event in this mechanism is induction of CYP2B enzymes with resulting liver hypertrophy and increased liver weights. These changes are followed by the second key event of liver cell proliferation progressing to liver tumors with chronic exposure. Consistent with this mechanism, the second key event of increased liver cell proliferation was only seen at the 7000 ppm concentration in the current study, which is the only concentration that produced liver tumors in the 18-month mouse feeding study.

Description of key information

Rat Uterotrophic Assay, Did not act as an estrogen receptor agonist; Reliability = 1

In vitro dopamine receptor binding assay, No activity, IC50> 10 µM; Reliability = 2

In Vitro testosterone and estrogen receptor binding assay, non-inhibitor, Reliability = 2

28-day mechanistic feeding study, mouse, liver changes occurred in association with the induction of cytochrome P450 enzyme activity and gene expression consistent with phenobarbital, Reliability = 2

Additional information

Rat uterine mode of action studies:

To evaluate the human relevance of the increased incidence of squamous cell carcinoma of the uterus/cervix in female rats observed in the long-term chronic toxicity/carcinogenicity study, two potential mechanisms were explored: estrogenicity and attenuation of circulating prolactin levels. Estrogen receptor agonists and chemicals that decrease circulating prolactin levels have both been shown to induce uterine tumors in rats. Estrogen is trophic in the uterus, and therefore increases uterine tumors via direct stimulation and proliferation of the uterine endometrium. Prolactin is luteotrophic in rodents, and not primates including humans, and thereby promotes progesterone production from the corpus luteum. Since progesterone antagonizes estrogenic stimulation of uterine growth, decreases in prolactin levels result in increased estrogenic stimulation of the uterus secondary to increased estrogen:progesterone ratio. In addition, decreased prolactin alters age-related reproductive changes in the rat, essentially delaying reproductive senescence and extending the duration of estrogen stimulation of the uterine endometrium (Harleman et al., (2012) Toxicologic Pathology 40:926-930).

In the studies performed with the test substance, the data from the in vitro estrogen receptor binding assay and the in vivo rat uterotrophic assay confirm that the test substance is not a direct estrogen agonist. In addition, the data from the uterotrophic assay indicates that the test substance decreases serum prolactin concentrations. Collectively, the data suggest that the induction of uterine tumors in the two-year rat feeding study occurred as a result of decreased prolactin, a mechanism not relevant to humans. While dopamine receptor agonists can result in reduced prolactin secretion by the pituitary, there was no evidence of dopamine receptor binding observed using in vitro receptor binding assays.

Mouse liver mode of action study:

 

The constitutive androstane receptor (CAR) is an important nuclear receptor involved in the regulation of cellular responses following exposure to many xenobiotics. Phenobarbital (PB) is a nongenotoxic prototypical pellCAR activator leading to increased gene expression and induction of hepatic cytochrome P450s (Cyps), hepatocellular hypertrophy and cell proliferation, and ultimately the development of liver tumors in mice and rats. However, there are species differences in the induction of liver tumors between rats and human by PB, including the lack of cell proliferation in cultured human hepatocytes leading to the conclusion that the mode of action for PB-induced rodent liver tumors is not plausible for humans. This conclusion is supported by epidemiological studies conducted on human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk (Elcombe et al., 2014).1

 

In the 18-month feeding study in mice, an increase in benign hepatocellular adenoma was observed in high dose males following exposure to test substance at 7000 ppm. Dose-dependent increases in liver weight, hepatocellular hypertrophy, and total cytochrome P450 levels were observed in a 28-day feeding study, with increased Cyp2B1/2, Cyp2E1 and Cyp4A1/2/3. Similar increases in liver weight and hepatocelluar hypertrophy were seen in a 90-day study. Therefore, a mechanistic study was undertaken to evaluate hepatic cytochrome P450 enzyme activity and gene expression, liver weight, liver microscopic pathology, and hepatocellular proliferation in male mice following dietary administration to the test substance for up to 28 days at dose levels of 200, 800, 2500 and 7000 ppm (the same levels used in the 18-month mouse study). A separate group of male mice was administered PB as a positive control. A dietary concentration of 7000 ppm resulted in hepatocellular hypertrophy, increased liver weight and hepatocelluar proliferation. The liver changes occurred in association with the induction of cytochrome P450 enzyme activity and gene expression consistent with PB, with the most pronounced changes occurring in Cyp2B10 gene expression (evaluated only in control and high dose at Days 3 and 8). The effects at 7000 ppm were observed at all time points evaluated in the study (Days 3, 8 and 29), except for the increase in cell proliferation which was only observed on Day 8. Mice administered 2500 ppm did not exhibit any increase in hepatocellular proliferation but did exhibit other liver effects (hepatocellular hypertrophy, increased liver weight, and minimal induction of cytochrome P450 enzyme activities), generally with lower magnitude or lower incidence relative to the 7000 ppm group. There were no biologically relevant effects at 200 or 800 ppm with respect to any parameter or time point evaluated.

 

The changes in the cytochrome P450 profile, along with the associated changes in the livers of the 7000 ppm male mice in this study, support a PB-like mechanism of action for liver tumor induction of male mice at 7000 ppm following chronic exposure at that dietary concentration. The first key event in this mechanism is induction of Cyp2B enzymes with resulting liver hypertrophy and increased liver weights. These changes are followed by the second key event of liver cell proliferation progressing to liver tumors with chronic exposure. Consistent with this mechanism, the second key event of increased liver cell proliferation was only seen at the 7000 ppm concentration, which is the only concentration considered to have produced a treatment-related increase in liver tumors in the 18-month mouse feeding study.