Registration Dossier
Registration Dossier
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EC number: 219-006-1 | CAS number: 2312-35-8
Specific investigations: other studies
Administrative data
Link to relevant study record(s)
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 April 2011 to 19 April 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 441
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1400
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vivo
- Endpoint addressed:
- other: Screening for androgen agonist/antagonist activity and 5α-reductase inhibition properties
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD®(SD) IGS
- Sex: Castrated males; surgical manipulation performed by the supplier
- Age at study initiation: Rats were postnatal day (PND; day of birth) 42 at surgery. Age at dose administration: PND 56/57.
- Weight at study initiation: 216.0 - 271.7 g
- Housing: Pre-allocation: 1 per cage; post allocation: 2 per cage. Cages were polycarbonate with micro-isolator top (23 cm wide by 44 cm long (1012 cm² area) and 21 cm high). Bedding was absorbent heat-treated hardwood bedding. Cages were changed twice per week.
- Diet: ad libitum
- Water: Reverse osmosis treated tap water (City of Durham, NC) ad libitum provided in glass water bottles with stainless steel sipper tubes (changed once per week).
- Acclimation period: Animals were acclimated in the study room for 8 days. Animals were received with surgical staples which were removed 4 days after receipt.
ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 23 °C
- Humidity: 30 to 64 %
- Photoperiod: 12/12 hour light/dark cycle
IN-LIFE DATES
From: 31 March 2011
To: 19 April 2011 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the testing facility in corn oil using silanised glassware at dose levels of 3, 9.4 and 30 mg/mL and dispensed into vials used daily during the study. Dose concentrations were adjusted in dose formulations to correct for purity of the test material. Dose formulations were stored at 1 to 10 °C.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was the vehicle used in oral gavage studies, including a dose range-finding study conducted by the testing facility
- Amount of vehicle (if gavage): Dose volume 5 mL/kg bodyweight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- DOSE FORMULATION ANALYSIS
Three samples (top, middle, and bottom) of the test material and TP control formulations were analysed in duplicate for concentration and homogeneity.
The acceptance criterion for concentration was within 15 % of the target concentration. Coefficient of variation less than 15 % was the acceptance criteria for homogeneity.
PREPARATION OF STOCK SOLUTIONS
A 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0562 g of the test material (0.0501 g as active ingredient) in a 50.0 mL volumetric flask and bringing it to volume with acetonitrile. Two secondary stock solutions (1.00 and 10.0 mg a.i./L) were prepared by placing 0.0500 and 0.500 mL, respectively, of the primary stock solution in individual 50.0 mL volumetric flasks and bringing each to volume with acetonitrile. These secondary stock solutions were used to prepare calibration standards for analysis of test material formulation samples.
A 1.00 mg/mL primary stock solution of TP was prepared by placing 0.0500 g of TP in a 50.0 mL volumetric flask and bringing it to volume with acetonitrile. A 1.00 mg/L secondary stock solution was prepared by placing 0.0500 mL of the primary stock solution in a 50.0 mL volumetric flask and bringing it to volume with acetonitrile. This stock solution was used to prepare calibration standards for analysis of the TP reference standards.
Stock solutions were stored in a refrigerator in glass amber bottles fitted with Teflon®-lined caps until use.
A 2000 mg/L dosing solution was prepared by placing 0.0200 g of TP in a 10.0 mL disposable glass vial and bringing it to volume with corn oil. This dosing stock solution was mixed using a magnetic stir plate and Teflon®-coated stir bar for approximately 45 minutes to ensure homogeneity and was used to prepare TP quality control samples.
PREPARATION OF CALIBRATION STANDARDS
Test material calibration standards were prepared in acetonitrile using the 1.00 and 10 mg a.i./L secondary stock solutions to yield concentrations of 0.0000100, 0.0000250, 0.0000500, 0.0000750, 0.000100 and 0.000200 mg a.i./mL.
TP calibration standards were prepared in acetonitrile using the 1.00 mg/L secondary stock solution to yield concentrations of 0.00100, 0.00200, 0.00500, 0.0100, 0.0250 and 0.0500 mg/L.
QUALITY CONTROL SAMPLE FORTIFICATION
Three test material quality control (QC) samples were individually prepared in corn oil.
For analysis of TP samples, three QC samples were prepared using the 2000 mg/L dosing stock solution.
SAMPLE DILUTION
Samples for analysis were vortexed for a minimum of 30 seconds after being allowed to equilibrate to room temperature. Duplicate aliquots of the test material formulation samples were removed and diluted first with hexane and then with acetonitrile.
Control samples were also analysed with each test material sample set and were diluted in the same fashion as the low concentration samples.
Duplicate aliquots of the TP samples were removed and diluted first with hexane and then with acetonitrile.
ANALYSIS
Samples were analysed for the test material and TP using liquid chromatography with mass spectrometry (LC/MS/MS).
Method validation studies were conducted prior to initiation of the definitive test and established average recoveries of 97.6 ± 5.90 % for the test material and 87.9 ± 1.78 % for TP from corn oil. Defined limits for acceptance of quality control sample performance were set at 85 to 115 %.
RESULTS
The mean measured concentrations for the test material samples prepared on the 04 April 2011 were 3.28, 10.4 and 29.9 mg a.i./mL for the low, mid and high dose groups, respectively. Analysis of QC samples resulted in recoveries ranging from 96.9 to 114 % (n = 3) of the nominal concentrations (1.50, 10.0 and 30.0 mg a.i./mL).
The mean measured concentrations for the test material samples prepared on the 07 April 2011 were 8.44 and 31.6 mg a.i./mL for the mid and high dose groups, respectively. Analysis of two of the three QC samples resulted in recoveries ranging from 85.5 to 90.9 % of the nominal concentrations (1.50, 10.0 and 30.0 mg a.i./mL). One of the three QC samples was measured at a 67.8 % recovery which was outside the range established during method validation. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors.
The mean measure concentration for the TP samples was 740 mg a.i./L. analysis of the QC samples resulted in recoveries ranging from 82.7 to 94.9 % (n = 3) of the nominal concentrations (400, 800 and 1200 mg/L). - Duration of treatment / exposure:
- 10 consecutive days (PND 56/57 through PND 65/66)
- Frequency of treatment:
- Once daily
- Post exposure period:
- Animals were euthanised 24 hours after the final dose
- Dose / conc.:
- 15 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 47 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 8 castrated males per dose
- Control animals:
- other: Yes; vehicle control, androgen agonist control (testosterone propionate, TP) and androgen antagonist control (flutamide, FT)
- Details on study design:
- Rats were allocated to one of eight designated dose groups. To evaluate the test material for androgen agonist properties, animals were administered one of two dose levels of the test material (47 or 150 mg/kg), the vehicle control, or the agonist reference material testosterone propionate (TP). To evaluate the test material for androgen antagonist properties animals were co-administered one of three dose levels of the test material (15, 47, or 150 mg/kg) or the antagonist flutamide with TP (0.4 mg/kg).
Animals were dosed for 10 consecutive days via oral gavage (test material and flutamide) and subcutaneous injection (TP). Changes in androgen dependent tissue weights were evaluated to determine the ability of the test material to act as an androgen agonist/antagonist or inhibitor of 5α-reductase.
ANIMAL ALLOCATION
The animals were assigned to a dose group using a procedure that allocated animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance (ANOVA).
DOSE ADMINISTRATION
Test material and FT dose formulations, along with the vehicle control (corn oil) were administered by oral gavage at a dosing volume of 5 mL/kg body weight. TP dose formulations were administered by subcutaneous injection into the dorsoscapular region at a dosing volume of 0.5 mL/kg body weight. In co-administered animals, oral gavage preceded subcutaneous injections.
Dosing occurred 24 hours (± 2 hours) from the previous dose. Dose volume was determined on individual animal daily body weight.
Group 4 animals were administered only TP subcutaneously. A separate study was conducted to compare body weights and androgen-dependent tissue weights of animals administered TP alone to animals co-administered TP and corn oil via gavage. Overall, this study showed no differences in androgen dependant tissue weights or body weights in animals administered TP only or co-administered TP and corn oil by oral gavage.
JUSTIFICATION OF DOSE LEVELS
The highest dose level took into consideration the LD50 and/or acute toxicity information in order to avoid death, severe suffering or distress in the animals and the second took into consideration available information on the doses used in subchronic and chronic studies. In general, the highest dose should not cause a reduction in the final body weight of the animals greater than 10 % of control weight. The highest dose should ensure animal survival without significant toxicity or distress to the animals after 10 consecutive days of administration up to a maximal dose of 1000 mg/kg/day.
The acute oral LD50 is 2800 mg/kg in male and female Sprague-Dawley rats. The NOEL for subchronic effects (13 weeks) was 100 ppm (5 mg/kg). In a chronic toxicity study, the test material was fed to male Sprague-Dawley rats at dietary concentrations of 0, 50, 80, 400 and 800 ppm (0, 2.38, 3.83, 19.24, and 38.87 mg/kg/day) for 24 months. Males displayed the greatest reduction in body weight gain at the 400 and 800 ppm dose levels during the initial weeks of the study. The magnitude of the decrease in body weight gain indicates that the MTD was exceeded in male rats at 800 ppm (estimated to be ~ 40 mg/kg/day).
In a range finder study, the test material was administered orally to male rats from PND 36 to 49 (14 days of dosing). After 10 days of dosing there were no observable clinical signs of toxicity in male rats, however body weight was reduced approximately 9 % in rats administered 150 mg/kg compared to control animals.
Based on the available data, a high dose of 150 mg/kg was selected to meet the requirements of achieving the highest dose which does not cause a reduction in the final body weight of the animals greater than 10 % of control weight, ensures animal survival, and is without significant toxicity or distress to the animals. - Examinations:
- IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity: Twice daily on weekdays, once daily on weekends.
- Clinical Observations: Observed within 2 days of arrival, again for allocation of animals to study groups, daily prior to dose administration and prior to euthanasia.
- Body Weights: Collected within 2 days of arrival, again for allocation of animals to study groups, daily prior to dose administration and prior to euthanasia.
TERMINATION
- Scheduled: Twenty four hours (± 2 hours) after the final dose administration, animals were humanely euthanised by carbon dioxide (CO2) asphyxiation with death confirmed by cervical dislocation in the same order as they were dosed.
- Tissue Collection: Gross observations of the tissues that were excised for tissue weights were recorded. In addition, preputial separation (PPS) was evaluated (inferred from necropsy records).
- Tissue Weights: The following tissues were excised, trimmed of excess adhering tissue and fat, and weighed to the nearest 0.1 mg: ventral prostate, seminal vesicles with coagulating gland with fluid, LABC (levator ani plus bulbocavernous muscle), Cowper’s glands (weighed as a pair) and the glans penis. - Positive control:
- Androgen agonist control (testosterone propionate, TP) and androgen antagonist control (flutamide, FT)
- Details on results:
- IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity
All animals survived to the scheduled humane euthanasia with no animals showing signs of moribundity.
- Clinical Observations
Androgen Agonist (Groups 1 to 3): One rat administered the test material (150 mg/kg) exhibited an ungroomed appearance prior to dosing on Day 6, but was clinically normal prior to and after this finding. No additional clinical observations were observed.
Androgen Antagonist (Groups 4 to 8): One rat co-administered TP and the test material (150 mg/kg) had clinical signs of an ungroomed appearance and oil fur around the anus prior to dosing on Day 5, while another animal in the same dose group was observed to have an ungroomed appearance on Day 6. Similar to the rat administered test material alone, the findings lasted a single day. No additional clinical observations were observed.
- Body Weights
Group 4 was used as the positive control and the negative control in the agonist and antagonist assays, respectively.
Androgen Agonist (Groups 1 to 4): Final body weight (84.9 % of control weight) and body weight gain of male rats administered 150 mg/kg test material were significantly decreased compared to the vehicle control group. Body weights of rats administered 47 mg/kg test material were not statistically different compared to the vehicle control. Administration of TP alone (positive control) resulted in a statistically significant increase in body weight gain compared to the vehicle control group, while final body weight between these two groups was not significantly different.
Androgen Antagonist (Groups 4 to 8): Co-administration of 150 mg/kg test material and TP caused a significant decrease in body weight (82.6 %) and body weight gain compared to the negative control group (TP alone). While body weight gain of rats co-administered 47 mg/kg test material and TP were statistically decreased compared to the negative control group, final body weights of rats were not. Body weights of rats administered 15 mg/kg test material were not statistically different compared to the negative control.
Co-administration of FT and TP did not affect body weights compared to the negative control group (TP alone).
NECROPSY PROCEDURES
- Gross Observations
Observations listed below of the 5 androgen-dependent tissues were made at necropsy, although observations relating to size (i.e. small) of tissues are not included.
Androgen Agonist (Groups 1 to 3): Animal 11 (47 mg/kg test material) - glans penis; prepuce cut to remove tissue (indicative of incomplete PPS).
Androgen Antagonist (Groups 4 to 8): Animal 27 (TP) - Cowper’s glands (right), red.
There was no statistical difference in incidence of PPS in the 47 mg/kg test material-administered group compared to the vehicle control group.
- Tissue Weights
Group 4 was used as the positive control and the negative control in the agonist and antagonist assays, respectively.
Tissue weights from one animal administered 150 mg/kg test material and one animal administered TP alone were removed prior to statistical analyses. The weights were excluded because the tissue weights were consistently identified as possible outliers (> 3 standard deviations).
Androgen Agonist (Groups 1 to 4): The weights of the 5 androgen dependent tissues were comparable between the vehicle control animals and animals administered test material. All five androgen-dependent tissues weights were significantly increased in the positive control (TP Group 4) as compared to vehicle control.
Androgen Antagonist (Groups 4 to 8): Co-administration of test material and TP resulted in a statistically significant decrease in the LABC and ventral prostate tissue weights at all dose levels compared to the negative control group. Seminal vesicle weights of rats co-administered 150 mg/kg test material and TP were significantly decreased compared to the negative control group. Glans penis and Cowper’s glands weights were not different from the control group. Administration of FT and TP significantly decreased all 5 androgen dependent tissue weights compared to the negative control group.
PERFORMANCE CRITERIA
Androgen Agonist (Groups 1 to 4): Calculated coefficient of variations (CVs) of the glans penis, Cowper’s glands and ventral prostate tissue weights from the vehicle control group and the CV of the Cowper’s glands from the rats administered 150 mg/kg test material were above the maximum allowable CVs for the agonist study. Mean glans penis and Cowper’s glands weights were decreased compared to controls suggesting that the high CVs would not impact the ability of the assay to detect androgen agonist activity. The remaining tissue CVs were within the allowable values.
Androgen Antagonist (Groups 4 to 8): Tissue CVs for the negative control (TP alone) and 150 mg/kg test material + TP dose group were within the allowable values. - Conclusions:
- Under the conditions of this Hershberger Assay, oral administration of the test material did not show any androgen agonist activity; however, the test material was positive in the antagonist assay, suggestive of 5α-reductase inhibition.
- Executive summary:
An Hershberger Bioassay was conducted to screen the test material for its androgen agonist/antagonist activity and 5α-reductase inhibition properties using a castrated rat model in accordance with the standardised guidelines US EPA OPPTS 890.1400 and OECD 441 under GLP conditions.
Sixty four castrated Sprague-Dawley rats were allocated to one of eight designated dose groups. To evaluate the test material for androgen agonist properties, animals were administered one of two dose levels of the test material (47 or 150 mg/kg), the vehicle control, or the agonist reference material testosterone propionate (TP). To evaluate the test material for androgen antagonist properties animals were co-administered one of three dose levels of the test material (15, 47, or 150 mg/kg) or the antagonist flutamide with TP (0.4 mg/kg).
Animals were dosed for 10 consecutive days via oral gavage (test material and flutamide) and subcutaneous injection (TP). Approximately twenty-four hours following the final dose administration, the animals were humanely euthanised; the glans penis, ventral prostate, levator ani plus bulbocavernous muscle (LABC), Cowper’s glands, and seminal vesicles with coagulating gland with fluid were excised and weighed.
Changes in androgen dependent tissue weights were evaluated to determine the ability of the test material to act as an androgen agonist/antagonist or inhibitor of 5α-reductase.
In evaluating the androgen agonist activity of the test material, final body weight and body weight gain of rats administered 150 mg/kg were statistically different as compared to the vehicle control (corn oil) animals. While final body weight was 85 % of control body weight, animals did not show any severe signs of toxicity or distress. Final body weight and body weight gain of animals administered 47 mg/kg test material were not statistically different than the vehicle control. There were no significant changes in androgen-dependent tissue weights compared to vehicle control animals at either test material dose level.
In the androgen antagonist assay, final body weight and body weight gain of animals co-administered 150 mg/kg test material and TP were statistically decreased compared to the negative control group. While final body weight in test material-administered rats was 83 % of the negative control body weight, animals did not show any severe signs of toxicity or distress. Body weight gain of animals co-administered 47 mg/kg test material and TP was significantly decreased, with no significant changes in final body weight. No changes in final body weight or weight gain were observed in animals co-administered 15 mg/kg test material and TP. Test material co-administered with TP caused significant decreases in LABC, ventral prostate, and seminal vesicles weights at 150 mg/kg compared to the negative control group. LABC and ventral prostate weights were also significantly decreased compared to the negative control weight when co-administered 15 and 47 mg/kg test material and TP. Glans penis and Cowper’s glands were not affected at any dose level of the test material.
Under the conditions of this Hershberger Assay which utilised the castrated rat model, oral administration of the test material did not show any androgen agonist activity; however, the test material did show antagonist activity or properties reflective of 5α-reductase inhibition.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 March 2011 to 29 July 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1450
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1500
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vivo
- Endpoint addressed:
- other: Identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal rat
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD®(SD) IGS
- Sex and number of animals: 25 time pregnant nulliparous dams (F0) were obtained at gestation day 8 to generate the F1 animals. Between postnatal days (PND) 3 and 5, litters with the same date of birth were standardised to 8 pups with equal numbers of males and females in the F1 generation.
- Age at study initiation: PND 22 (females); PND 23 (males) where PND 0 is the date of birth
- Weight at study initiation: 44.7 - 63.6 g (females); 49.6 - 71.2 g (males)
- Housing: F0: 1 per cage (with litter); F1: 2 per cage. Cages were polycarbonate with micro-isolator top (23 cm wide by 44 cm long (1012 cm² area) and 21 cm high). Bedding was absorbent heat-treated hardwood bedding. Cages were changed once per week without a litter and twice per week with the litter for F0 animals. Cages were changed twice per week for the F1 animals.
- Diet: ad libitum
- Water: Reverse osmosis treated tap water (City of Durham, NC) ad libitum provided in glass water bottles with stainless steel sipper tubes (changed once per week).
- Acclimation period: From PND 0 to PND 21/22
ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23 °C
- Humidity: 09 to 63 % (The humidity was within the acceptable range (30 to 70 %) during the conduct of the study except for three occasions; these deviations occurred prior to time-pregnant dams giving birth and not during dose administration.
- Photoperiod: 14/10 hour light/dark cycle
IN-LIFE DATES
From: 17 February 2011
To: 29 July 2011 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the testing facility twice in corn oil using silanised glassware at concentrations of 12.5 and 25 mg/mL and dispensed into vials to be used daily during the study. Dose concentrations were adjusted in dose formulations to correct for purity of the test material. Dose formulations were stored at room temperature.
VEHICLE
- Amount of vehicle (if gavage): Dose volume 5 mL/kg bodyweight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- DOSE FORMULATION ANALYSIS
Three samples (top, middle, and bottom) of the test material formulations prepared on the 22 March 2011 and the 7 and 8 April 2011 were analysed in duplicate for concentration and homogeneity.
The acceptance criterion for concentration was within 15 % of the target concentration. Coefficient of variation less than 15 % was the acceptance criteria for homogeneity.
Samples were received for analysis as follows:
- 23 March 2011: Nominal concentrations of 12.5 and 25 mg a.i./mL and a control
- 05 April 2011: Nominal concentrations of 12.5 and 25 mg a.i./mL
- 08 April 2011: Nominal concentrations of 12.5 and 19 mg a.i./mL
- 12 April 2011: Nominal concentration of 25 mg a.i./mL and a control
PREPARATION OF STOCK SOLUTIONS
Prior to the first analysis and again prior to analysis of the 5 April samples, a 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0561 to 0.0562 g of the test material (0.0500 to 0.0501 g as active ingredient) in a 50.0 mL volumetric flask and bringing it to volume with acetonitrile. Two secondary stock solutions (1.00 and 10.0 mg a.i./L) were prepared by placing 0.0500 and 0.500 mL, respectively, of the primary stock solution in individual 50.0 mL volumetric flasks and bringing each to volume with acetonitrile. These secondary stock solutions were used to prepare calibration standards.
Stock solutions were stored in a refrigerator in glass amber bottles fitted with Teflon®-lined caps until use.
PREPARATION OF CALIBRATION STANDARDS
Test material calibration standards were prepared in acetonitrile using the 1.00 and 10 mg a.i./L secondary stock solutions to yield concentrations of 0.0000100, 0.0000250, 0.0000500, 0.0000750, 0.000100 and 0.000200 mg a.i./mL.
QUALITY CONTROL SAMPLE FORTIFICATION
Three test material quality control (QC) samples were individually prepared in corn oil.
SAMPLE DILUTION
The samples received on the 23 March (one control and two samples from each dose level) were analysed in duplicate for concentration and homogeneity. On the 5 April, three samples from the 12.5 mg a.i./mL and 6 samples from the 25 mg a.i./mL concentration levels were received and analysed I duplicate for concentration only.
The samples received on the 8 and 12 April were analysed in duplicate for verification of concentration and homogeneity.
Large volume samples were mixed for at least 45 minutes using magnetic stir plates and Teflon®-coated stir bars prior to dilution to allow equilibration to room temperature. Smaller aliquots were vortexed for a minimum of 30 seconds after being allowed to equilibrate to room temperature. Samples were removed and diluted first with hexane and then with acetonitrile.
Control samples were also analysed with each test material sample set and were diluted in the same fashion as the low concentration samples.
ANALYSIS
Samples were analysed for the test material using liquid chromatography with mass spectrometry (LC/MS/MS).
Method validation studies were conducted prior to initiation of the definitive test and established an average recovery of 97.6 ± 5.90 % for the test material from corn oil. Defined limits for acceptance of quality control sample performance were set at 85 to 115 %.
RESULTS
The mean measured concentrations for the test material samples received on the 23 March 2011 were 13.7 and 25.7 mg a.i./mL for the low and high concentration samples (12.5 and 25 mg a.i./mL), respectively. Analysis of five of the six QC samples resulted in recoveries ranging from 95.5 to 111 % of the nominal fortified concentrations (1.50, 10.0 and 30.0 mg a.i./mL). One of the six QC samples was measured at a 123 % recovery which was outside the range established during method validation. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors.
The mean measured concentrations for the test material samples received on the 05 April 2011 were 12.2 and 24.5 mg a.i./mL for the low and high concentration samples, respectively. Analysis of QC samples resulted in recoveries ranging from 91.7 to 116 % (N = 3) of the nominal fortified concentrations (1.50, 10.0 and 30.0 mg a.i./mL).
The mean measured concentration for the 12.5 mg a.i./mL test material samples received on the 8 April 2011 was 12.4 mg a.i./mL for the low concentration samples (12.5 mg a.i./mL). Analysis of two of the three QC samples resulted in recoveries ranging from 86.0 to 91.5 % of the nominal fortified concentrations (1.50, 10.0 and 30.0 mg a.i./mL). One of the three QC samples was measured at a 79.2 % recovery which was just slightly outside the range established during method validation. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors. The 19 mg a.i./mL samples also received on this date were analysed along with those received on the 12 April.
The mean measured concentrations for the 19 mg a.i./mL test material samples received on the 08 April 2011 and the 25 mg a.i./mL samples received on the 12 April were 18.6 and 27.2 mg a.i./mL for the mid and high concentration samples, respectively. Analysis of QC samples resulted in recoveries ranging from 101 to 114 % (N = 6) of the nominal fortified concentrations (1.50, 10.0 and 30.0 mg a.i./mL). - Duration of treatment / exposure:
- The dose formulations were administered for 21/22 (females, PND 22 through 42 or 43) or 31/32 (males, PND 23 through 53 or 54) consecutive days.
- Frequency of treatment:
- Once daily
- Post exposure period:
- Animals were euthanised at least 2 hours after the final dose
- Dose / conc.:
- 62.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 125 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 16 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The purpose of the assay was to identify the potential of the test material to interact with the endocrine system by identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal female or male rat.
ANIMAL ALLOCATION
The animals were assigned to a dose group using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance [ANOVA].
DOSE ADMINISTRATION
Dosing occurred 24 hours (± 2 hours) from the previous dose. Dose volume was determined on individual animal daily body weight.
JUSTIFICATION OF DOSE LEVELS
High dose levels were expected to meet the maximum tolerated dose (MTD) level. The second dose was selected to be one-half of the high dose level.
A 14-day dose range finding study was conducted at the testing facility to select a dose to approach, but not exceed the MTD. Three rats per sex were administered the test material at dose levels of 0, 50, 75, 100, 125 and 150 mg/kg for 10 days.
Female rats administered 150 mg/kg exhibited a statistically significant decrease in body weight gain and a final body weight that was decreased 21 % compared to vehicle controls. Body weight gain in females administered 50, 75, 100 and 125 mg/kg was not significantly different to vehicle controls.
Males administered 150 mg/kg demonstrated a statistically significant decrease in body weight gain and a 10 % decrease in final body weight compared to vehicle control animals however; no significant reduction in body weight gain was observed for males administered 50, 75, 100 and 125 mg/kg. While no adverse clinical observations were noted at any dose level, in either male or female rats, it was not clear that the animals administered 150 mg/kg would tolerate an additional 14 days of dose administration.
A high dose of 125 mg/kg was selected for female and male rats to satisfy the requirements of achieving a high dose that approached, but did not exceed the MTD level. - Examinations:
- IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity
Twice daily on weekdays, once daily on weekends/holidays
- Clinical Observations
F0 animals were observed within 2 days of arrival. F1 animals were observed for allocation of animals to study groups, daily prior to dose administration and prior to euthanasia.
- Body Weights
Body weights were collected on F0 dams within 2 days of arrival. Body weights were collected on F1 animals weekly following birth, for allocation of animals to study groups, daily prior to dose administration and prior to euthanasia.
- Vaginal Opening
Beginning on PND 22, F1 females were examined daily following dosing for vaginal opening (VO); VO was recorded as either VO not initiated, appearance of a pin hole, vaginal thread or complete VO.
- Oestrous Cyclicity
Beginning on the day of complete VO through euthanasia, vaginal smears were prepared daily, evaluated and classified as either dioestrus, pro-oestrus, or oestrus; metoestrus was classified as dioestrus. The overall cycling pattern for each female was characterized as regular, irregular, or not cycling. Regular cycling was defined as having recurring 4 to 5 day cycles, while irregular cycling was defined as having cycles with dioestrus for a period > 3 days or oestrus ≥3 days. Animals were not considered to be cycling if there was ≥3 consecutive days of oestrus or ≥5 consecutive days of dioestrus. Mean cycle length was defined as the number of days from one dioestrus to the next dioestrus. Incomplete cycles were not used in calculating mean cycle length.
In instances where the time between VO and humane euthanasia did not allow observation of more than 1 complete cycle, classification was based on data available and the assumption that animals were cycling regularly if the partial data fit the definition, and cycling irregularly if the study ends without being able to distinguish between irregular and not cycling.
- Preputial Separation
Beginning on PND 30, F1 males were examined daily following dosing for preputial separation (PPS); PPS was recorded as either progression not initiated, partial separation, a persistent thread of tissue between the glans and prepuce or complete PPS.
TERMINATION
- Moribunds/Unscheduled
Beginning on the first day of dose administration, any animals found moribund or dead were necropsied and cause of death was determined and recorded, if possible. Moribund animals were euthanised by carbon dioxide (CO2) inhalation and death confirmed by cervical dislocation.
- Scheduled
Animals were moved to the necropsy room holding room at least 2 hours before euthanasia. At least 2 hours after the final dose administration animals were humanely euthanised by decapitation, in the same order as they were dosed.
- Blood Collection
Prior to tissue collection, trunk blood was collected. Blood was collected in a glass serum separator tube and stored on wet ice until centrifugation. Blood was centrifuged at 3000 x g for 30 minutes at 4 °C. Serum was aliquoted into siliconised microcentrifuge tubes. Serum prepared from male rats was found to be severely lipemic and were re-centrifuged at 10 000 x g for 30 minutes at 4 °C. All serum samples were stored at or below -70 °C.
- Tissue Collection
Gross observations of the tissues that were excised for tissue weights were recorded.
- Tissue Weights
From the F1 females, the following tissues were excised, trimmed of excess adhering tissue and fat, and weights recorded to the nearest 0.0001 g: adrenals (paired), kidneys (paired), liver, thyroid (post fixation*), ovaries (paired), uterus (with and without fluid) and pituitary.
From the F1 males, the following tissues were excised, trimmed of excess adhering tissue and fat, and weights recorded to the nearest 0.0001 g: adrenals (paired), kidneys (paired), liver, thyroid (post fixation*), ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating gland (with and without fluid), levator ani plus bulbocavernous muscle complex, epididymides (left and right separately), testes (left and right separately) and pituitary.
*The thyroid with attached trachea was fixed in 10 % neutral buffered formalin (NBF) for at least 24 hours. The thyroid was then dissected from the trachea, blotted, weighed to the nearest 0.00001 g, and transferred to 70 % histology grade alcohol.
- Hormone and Clinical Chemistry Concentrations
T4 and TSH (male and female sera) and testosterone (T) (male sera) was measured in serum using radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with multiple quality control samples.
Serum samples from both sexes were analysed for creatinine, blood urea nitrogen, sorbital dehydrogenase, sodium, potassium, chloride, calcium, phosphorous, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, and total bilirubin.
- Histopathology
Uterus (without fluid), left ovary, left kidney, and thyroid were fixed in 10 % NBF for at least 24 hours, transferred to 70 % histology grade alcohol, histologically processed, embedded, sectioned, and stained with haematoxylin and eosin (H&E). Five random sections of the ovary and two 2 serial sections of thyroid gland taken from approximately halfway into the tissue were evaluated.
Left testis and left epididymis was placed in Bouin’s fixative for 18 to 24 hours and then washed in 70 % histology grade alcohol. After fixation, tissues were transferred to 70 % histology grade alcohol, histologically processed, embedded, sectioned, and stained with H&E.
Histologically processed tissues were microscopically evaluated by the study pathologist. Evaluation of each ovarian section included: follicular development (including presence/absence of tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum development, and changes in number of both primary and atretic follicles) in addition to any abnormalities/lesions, such as ovarian atrophy. Two sections of each of the 2 lobes of the thyroid were examined and evaluated for follicular cell height and colloid area using a five-point grading scale and the thyroids were evaluated relative to photomicrographs supplied by the US EPA. - Details on results:
- FEMALES
IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity
All female rats administered 0 (vehicle control) or 62.5 mg/kg test material (Groups 1 and 2) survived to scheduled euthanasia with no animals showing signs of moribundity. Two animals administered 125 mg/kg test material (Group 3) did not survive to scheduled euthanasia; one rat was moribund and euthanised on study day 15 (PND 36), and one rat was found dead on day 19 (PND 40). There was no gross evidence of dosing error present during necropsy of either rat administered 125 mg/kg test material.
- Clinical Observations
Clinical observations (24 hours) were performed post dose administration. No clinical signs of toxicity were observed 24 hours after dose administration in any female rats administered 0 (vehicle control) or 62.5 mg/kg test material (Groups 1 and 2).
In one animal administered 125 mg/kg test material (Group 3) piloerection, diarrhoea, thin, hunched posture, and/or a distended abdomen were noted 24 hours after dose administration between days 10 and 15 (PND 31 to 36) prior to being euthanised on PND 36 as described above. All other rats appeared normal throughout the study.
- Body Weight
No significant change in mean final body weight or body weight gain was observed in females administered 62.5 mg/kg test material (Group 2) compared to the vehicle control group (Group 1). In females administered 125 mg/kg test material (Group 3), mean final body weight (86 % of control group) and body weight gain were significantly reduced compared to the vehicle control group.
- Vaginal Opening
No significant difference was noted in the age or body weight of female rats at complete VO for animals administered 62.5 mg/kg test material (Group 2) compared to the vehicle control group (Group 1). In animals administered 125 mg/kg test material (Group 3), mean age of complete and initiation of VO was significantly delayed compared to the vehicle control group (Group 1). The mean age for complete VO in animals administered 125 mg/kg test material (Group 3) was PND 41.1 ± 2.8 compared to PND 34.3 ± 4.0 for the vehicle control animals. Body weight of animals administered 125 mg/kg test material (Group 3) was significantly increased at complete VO compared to the vehicle control group (Group 1).
- Oestrous Cyclicity
Administration of 62.5 mg/kg test material (Group 2) did not change the onset of first oestrus compared to the vehicle control group (Group 1), however administration of 125 mg/kg test material significantly delayed day of first oestrus from PND 34.7 (vehicle control group) to PND 39.1. Animals administered 0 (vehicle control), 62.5 and 125 mg/kg test material exhibited similar cycle lengths and regularity.
TERMINATION
- Gross Observations
A clear 2 x 2 mm cyst was observed on the left kidney of one animal administered 125 mg/kg test material (Group 3). No other gross observations were noted for other female rats surviving to the scheduled termination.
- Tissue Weights
Administration of 125 mg/kg test material (Group 3) significantly increased relative, but not absolute, liver weights compared to the vehicle control group (Group 1).
Administration of 62.5 or 125 mg/kg test material (Group 2 and 3) significantly increased relative, but not absolute, kidney weights compared to the vehicle control group (Group 1).
Administration of 125 mg/kg (Group 3) test material decreased absolute and relative pituitary weight and administration of 62.5 mg/kg test material (Group 2) decreased absolute pituitary weight compared to the vehicle control group (Group 1). Ovaries and uterine (wet and blotted) weights were significantly decreased following administration of 125 mg/kg test material (Group 3), but not 62.5 mg/kg test material (Group 2), compared to the vehicle control group (Group 1). Adrenal and thyroid weights were not changed following administration of test material compared to the vehicle control group (Group 1).
- Hormone and Clinical Chemistry Analysis
Administration of 125 (Group 3), but not 62.5 mg/kg test material (Group 2), significantly increased serum T4 concentrations compared to the vehicle control group (Group 1). No significant changes in serum TSH concentrations were detected following administration of either 62.5 or 125 mg/kg test material (Groups 2 and 3) compared to the vehicle control group (Group 1).
In female rats administered 62.5 or 125 mg/kg test material (Groups 2 and 3) serum sodium, chloride, and sorbital dehydrogenase concentrations were significantly increased compared to controls, whereas alkaline phosphatase and total serum protein concentrations were significantly decreased compared to the vehicle control group (Group 1). Significant changes were also noted for serum concentrations of calcium (decreased in Group 2; 62.5 mg/kg test material), total bilirubin (increased in Group 3; 125 mg/kg test material) and albumin (decreased in Group 3; 125 mg/kg test material) compared to the vehicle control group (Group 1). There were no changes in phosphorous, alkaline aminotransferase, alanine aminotransferase, gamma glutamyl transferase, blood urea nitrogen, and creatinine levels following administration of the test material.
- Histopathology
Administration of test material for 21/22 days to female rats resulted in histopathological changes in the uterus, ovary, and thyroid gland. Administration of 125 mg/kg (Group 3) caused a significant increase in the thyroid gland follicular epithelial cell height. A corresponding significant decrease in the colloid area of thyroid follicles occurred in female rats following administration of 62.5 mg/kg or 125 mg/kg test material (Groups 2 and 3) compared to control rats.
Histologic alterations in the ovary attributed to test material administration included increases in the number of small ovarian follicles and follicular cysts, and a decrease in the number of corpora lutea compared to the control animals. The incidence and severity of interstitial cell hyperplasia in the ovaries were greater in rats administered 125 mg/kg (Group 3) than controls. Uterine atrophy was present in 3 of 14 rats (21 %) that were administered 125 mg/kg test material (Group 3), whereas no comparable lesions were found in rats administered 62.5 mg/kg test material (Group 2) or controls.
- Performance Criteria
Data generated in this study from the vehicle control rats were within the US EPA performance criteria with the following exceptions: final body weight, age and weight at VO, kidney and liver weight CVs were slightly above the acceptable range. Mean adrenal weights were below the acceptable mean range.
MALES
IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity
All male rats administered 0 (vehicle control), 62.5 and 125 mg/kg test material (Groups 4 to 6) survived to scheduled termination with none showing signs of moribundity.
- Clinical Observations
Clinical observations (24 hours) were performed post dose administration. Red ocular discharge was observed in one rat administered the vehicle control (Group 4) on PND 38. Salivation prior to dosing was seen in one rat administered 62.5 mg/kg test material (Group 5) prior to dosing on Day 16. One rat administered 125 mg/kg test material (Group 6) was noted as being thin 24 hours after dose administration from study day 16 (PND 38) until scheduled euthanasia; in addition this rat was noted as ungroomed on day 31 (PND 53). Bleeding from the nose was observed in one rat administered 125 mg/kg test material (Group 6) on PND 46 and another rat in the same group was noted to have alopecia on the throat on PND 53. Two animals administered 125 mg/kg test material (Group 6) were observed to be thin; one rat PND 51 and 52 and one rat PND 54. All other rats were observed as normal.
- Body Weight
No significant change in mean final body weight or body weight gain was observed in males administered 62.5 mg/kg test material (Group 5) compared to the vehicle control group (Group 4). In males administered 125 mg/kg test material (Group 6) mean final body weight (77 % of control group) and body weight gain were significantly reduced compared to the vehicle control group.
- Preputial Separation (PPS)
No significant difference was noted in the age or body weight of males at complete or partial PPS for animals administered 62.5 mg/kg test material (Group 5) compared to the vehicle control group (Group 4). In animals administered 125 mg/kg test material (Group 6), mean age of complete and partial PPS was significantly delayed compared to the vehicle control group (Group 4). The mean age of complete PPS in Group 4 animals was PND 45.2 ± 2.3 compared to PND 50.3 ± 3.1 for Group 6 animals. Body weight of animals at complete PPS was not significantly different in test material administered animals (Groups 5 and 6) compared to vehicle control group (Group 4).
TERMINATION
- Gross Observations
No gross observations were noted in male rats surviving to the scheduled termination.
- Tissue Weights
Administration of 125 mg/kg test material (Group 6) significantly decreased absolute kidney weight and significantly increased relative kidney weights, while administration of 62.5 mg/kg test material (Group 5) significantly decreased relative kidney weights compared to the vehicle control group (Group 4). Administration of 125 mg/kg test material (Group 6) significantly decreased absolute liver and adrenal weights, and significantly increased relative liver and adrenal weights compared to the vehicle control group (Group 4). Relative, but not absolute, pituitary weights were significantly increased following administration of 62.5 or 125 mg/kg test material (Groups 5 and 6) compared to the vehicle control group (Group 4).
Administration of 125 mg/kg test material (Group 6) significantly decreased seminal vesicle plus coagulating gland with and without fluid, ventral prostate, dorso-lateral prostate, LABC, and thyroid gland weights compared to the vehicle control group (Group 4). Epididymides and testes weights were not found to be affected following administration of test material when compared to the weights of these tissues in the vehicle control group (Group 4).
- Hormone and Clinical Chemistry Concentrations
The serum concentration of T4 was significantly increased and testosterone was significantly decreased in rats administered 62.5 or 125 mg/kg test material (Groups 5 and 6) compared to the vehicle control group (Group 4). No changes in serum TSH concentration were found in males administered either dose level of test material.
Chloride and alkaline phosphatase serum concentrations in rats administered 62.5 and 125 mg/kg test material (Groups 5 and 6) were significantly decreased compared to the vehicle control group (Groups 4). Significant changes were also noted for serum concentrations of alanine aminotransferase (decreased in Group 5), sorbital dehydrogenase (increased in Group 6) and total protein (decreased in Group 6) compared to the vehicle control group (Group 4). There were no changes in sodium, potassium, calcium, phosphorous, aspartate aminotransferase, gamma glutamyl transferase, blood urea nitrogen, creatinine, and total bilirubin levels following administration of test material.
- Histopathology
Administration of the test material for 31/32 days to male rats resulted in histopathological changes in the thyroid gland and kidneys. In the thyroid gland, a significant increase in follicular cell height and decrease in the follicular colloid area was observed at both test material dose levels (Groups 5 and 6).
In the kidney, tubular epithelial cell karyomegaly was evident in 81.3 and 100 % of rats administered 62.5 or 125 mg/kg test material, respectively (Groups 5 and 6). Hyperplasia and hypertrophy of tubular epithelial cells was observed in 13 and 100 % of rats administered 62.5 or 125 mg/kg test material, respectively (Groups 5 and 6). Hyaline droplets were observed in 88 and 94 % of rats administered 62.5 or 125 mg/kg test material, respectively (Groups 5 and 6).
- Performance Criteria
Data generated in this study from the vehicle control rats were within the US EPA performance criteria with the following exceptions: final body weight, weight at PPS, and pituitary weight CVs were above the acceptable range. Mean kidney weight was below the acceptable mean range. TSH serum concentration was below the acceptable mean range and the CV was above the acceptable range. - Conclusions:
- Under the conditions of this study, administration of the test material resulted in changes in endpoints that suggest an effect on pubertal development and thyroid function (i.e. histopathological changes) at a dose level (125 mg/kg) that does, and does not (62.5 mg/kg), exceed the maximum tolerated dose (MTD).
- Executive summary:
An assay was conducted to identify the potential of test material to interact with the endocrine system by identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal female or male rat in accordance with the standardised guidelines US EPA OPPTS 890.1450 and 890.1500, respectively, under GLP conditions.
Groups of 16 Sprague-Dawley rats per dose per sex were orally administered the test material (62.5 or 125 mg/kg) or corn oil (vehicle control) for 21/22 (females) or 31/32 (males) days. Approximately two hours following the final dose administration, the animals were humanely euthanised.
- Females
Administration of the test material to female rats affected pubertal development by delaying vaginal opening, oestrus onset (with no change in cyclicity), and decreasing ovarian and uterine weights with changes in histopathology that suggest the test material may inhibit steroidogenesis, or, based on the significant decreases in pituitary weight (relative and absolute), disrupt the hypothalamic-pituitary-gonadal axis. After 21/22 days of test material (125 mg/kg) administration there was an increase in circulating T4 concentration with no corresponding change in TSH. Administration of the test material did not result in a change in thyroid gland weight compared to the control group, however histopathological changes, including an increase in follicular cell height (125 mg/kg) and a decrease in colloid area (62.5 and 125 mg/kg), were significant compared to the control group.
The majority of the changes that were measured in this study were detected following a dose of test material (125 mg/kg) that although did not cause adverse toxicity in the animals, resulted in a greater than 10 % (~14 %) decrease in body weight compared to the control group. Changes in development detected in this assay may be attributed to body weight loss and not a direct effect of the chemical. Since this study suggests that interaction with the endocrine system occurs at a dose level that causes greater than a 10 % body weight loss compared to controls, it will require information from other Tier 1 assays for a complete interpretation of the significant findings.
- Males
Administration of the test material to male rats caused changes in pubertal development including delayed PPS, decreases in weights in most, but not all, of the androgen dependent tissues (i.e. seminal vesicle plus coagulating gland with and without fluid, ventral prostate, dorso-lateral prostate, and LABC) along with a decrease in serum testosterone (at 62.5 and 125 mg/kg test material) that suggest test material may inhibit steroidogenesis, or disrupt the hypothalamic-pituitary- gonadal axis. After 31/32 days of test material (62.5 or 125 mg/kg) administration there was an increase in circulating T4 concentration with no corresponding change in TSH. There was a significant decrease in thyroid gland weight in animals administered 125 mg/kg test material compared to the control group, with histopathological changes, including an increase in follicular cell height with a decrease in colloid area, following administration of either 62.5 or 125 mg/kg test material.
The majority, but not all of the endpoint changes that were measured in this study were detected following a dose of test material that caused a greater than 10 % (~23 %) decrease in body weight compared to the control group. These changes may be attributed to body weight loss and not a direct effect of the chemical. Since this study suggests that interaction with the endocrine system occurs at a dose level that causes more than approximately a 10 % body weight loss compared to controls, it will require information from other Tier 1 assays for a complete interpretation of the significant findings.
Overall, administration of the test material to intact juvenile/peripubertal female and male rats resulted in changes in endpoints that suggest an effect on pubertal development and thyroid function (i.e. histopathological changes) at a dose level (125 mg/kg) that does, and does not (62.5 mg/kg), exceed the maximum tolerated dose (MTD).
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 April 2011 to 05 April 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 440
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1600
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vivo
- Endpoint addressed:
- other: Uterotrophic assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD®(SD) IGS
- Sex: Ovariectomised females; surgical manipulation performed by the supplier
- Age at study initiation: Rats were postnatal day (PND; day of birth) 49 at arrival. Age at dose administration: PND 61.
- Weight at study initiation: 223.0 - 288.5 g
- Housing: Pre-allocation: 1 per cage; post allocation: 2 per cage. Cages were polycarbonate with micro-isolator top (23 cm wide by 44 cm long (1012 cm² area) and 21 cm high). Bedding was absorbent heat-treated hardwood bedding. Cages were changed twice per week.
- Diet: ad libitum
- Water: Reverse osmosis treated tap water (City of Durham, NC) ad libitum provided in glass water bottles with stainless steel sipper tubes (changed once per week).
- Acclimation period: Animals were acclimated in the study room for 12 days.
ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 23 °C
- Humidity: 30 to 49 %
- Photoperiod: 12/12 hour light/dark cycle
IN-LIFE DATES
From: 21 March 2011
To: 05 April 2011 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the testing facility in corn oil using silanised glassware at dose levels of 25 and 80 mg/mL and aliquoted into vials used daily during the study. Dose concentrations were adjusted in dose formulations to correct for purity of the test material. Dose formulations were stored at 1 to 10 °C.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was the vehicle used in oral gavage studies, including a dose range-finding study conducted by the testing facility
- Amount of vehicle (if gavage): Dose volume 5 mL/kg bodyweight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- DOSE FORMULATION ANALYSIS
Three samples (top, middle, and bottom) of the test material and TP control formulations were analysed in duplicate for concentration and homogeneity.
The acceptance criterion for concentration was within 15 % of the target concentration. Coefficient of variation less than 15 % was the acceptance criteria for homogeneity.
PREPARATION OF STOCK SOLUTIONS
A 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0561 g of the test material (0.0500 g as active ingredient) in a 50.0 mL volumetric flask and bringing it to volume with acetonitrile. Two secondary stock solutions (1.00 and 10.0 mg a.i./L) were prepared by placing 0.0500 and 0.500 mL, respectively, of the primary stock solution in individual 50.0 mL volumetric flasks and bringing each to volume with acetonitrile. These secondary stock solutions were used to prepare calibration standards.
Stock solutions were stored in a refrigerator in glass amber bottles fitted with Teflon®-lined caps until use.
PREPARATION OF CALIBRATION STANDARDS
Test material calibration standards were prepared in acetonitrile using the 1.00 and 10 mg a.i./L secondary stock solutions to yield concentrations of 0.0000100, 0.0000250, 0.0000500, 0.0000750, 0.000100 and 0.000200 mg a.i./mL.
QUALITY CONTROL SAMPLE FORTIFICATION
Three test material quality control (QC) samples were individually prepared in corn oil.
SAMPLE DILUTION
Samples were mixed for at least 45 minutes using magnetic stir plates and Teflon®-coated stir bars prior to dilution to allow equilibration to room temperature. On 23 March 2011 one sample was received from each dose level and the testing facility sampled the top, middle and bottom and analysed in duplicate for verification of concentration and homogeneity. On 31 March 2011, three aliquots (sampled from the top, middle and bottom of each dose formulation) were received and duplicate samples analysed for verification of concentration and homogeneity.
Samples were removed and diluted first with hexane and then with acetonitrile.
Control samples were also analysed with each test material sample set and were diluted in the same fashion as the low concentration samples.
ANALYSIS
Samples were analysed for the test material using liquid chromatography with mass spectrometry (LC/MS/MS).
Method validation studies were conducted prior to initiation of the definitive test and established an average recovery of 97.6 ± 5.90 % for the test material from corn oil. Defined limits for acceptance of quality control sample performance were set at 85 to 115 %.
RESULTS
The mean measured concentrations for the test material samples received on the 23 March 2011 were 25.9 and 87.2 mg a.i./mL for the low and high dose groups, respectively. Analysis of QC samples resulted in recoveries ranging from 88.7 to 104 % (n = 3) of the nominal concentrations (1.50, 10.0 and 30.0 mg a.i./mL).
The mean measured concentrations for the test material samples received on the 31 March 2011 were 22.4 and 81.4 mg a.i./mL for the low and high concentration samples, respectively. Analysis of five of the six QC samples resulted in recoveries ranging from 87.3 to 108 % of the nominal concentrations (1.50, 10.0 and 30.0 mg a.i./mL). One of the six QC samples was measured at a 77.4 % recovery which was just slightly outside the range established during method validation. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors. - Duration of treatment / exposure:
- 3 days
- Frequency of treatment:
- Once daily
- Post exposure period:
- Animals were euthanised 24 hours after the final dose
- Dose / conc.:
- 125 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 400 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 8 females per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The purpose of the assay was to evaluate the test material for its oestrogenic effects utilising the ovariectomised rat model in the Uterotrophic Assay
ANIMAL ALLOCATION
The animals were assigned to a dose group using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance [ANOVA].
DOSE ADMINISTRATION
Dosing occurred 24 hours (± 2 hours) from the previous dose. Dose volume was determined on individual animal daily body weight.
JUSTIFICATION OF DOSE LEVELS
The highest dose took into consideration the LD50 and/or acute toxicity information in order to avoid death, severe suffering or distress in the animals. The highest dose represents the maximum tolerated dose (MTD). The second dose level was one half log unit smaller.
The acute oral LD50 for the test material is 2800 mg/kg in male and female Sprague-Dawley rats. In a range-finding study conducted at the testing facility, the test material was orally administered to immature female rats, (50 to 150 mg/kg in 25 mg/kg increments) from PND 22 to 35 (14 days of dosing). After 3 days of dosing there were no clinical signs of toxicity or change in body weights in the animals dosed with test material compared to the vehicle controls.
In a second dose range finding study conducted at the testing facility, adult female rats were administered test material from 0 to 1000 mg/kg with 200 mg/kg increments for 3 consecutive days. Doses of 800 and 1000 mg/kg resulted in mortality and moribundity. Two animals administered 600 mg/kg were found dead the day following the last dose. Animals administered 400 mg/kg survived until the scheduled euthanasia. Body weights were ~88 % of the concurrent controls and clinical observations included an ungroomed appearance and a greasy rectal area which was first noted prior to the second daily dose and continued until euthanasia. Fur discoloration of the head in one animal was observed prior to the third dose and in all animals the following morning.
Based on the above mentioned studies, the current test guideline dosing regimen (3 days of dosing), and animal model, a high dose of 400 mg/kg test material was selected to meet the requirements of achieving a MTD). - Examinations:
- IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity: Twice daily on weekdays, once daily on weekends.
- Clinical Observations: Observed within 2 days of arrival, again for allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia.
- Cage-side Observation: Observed 3 hours (± 30 minutes) following dose administration.
- Body Weights: Collected within 2 days of arrival, again for allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia.
TERMINATION
- Scheduled: Twenty four hours (± 2 hours) after the final dose administration, animals were humanely euthanised by carbon dioxide (CO2) asphyxiation with death confirmed by cervical dislocation in the same order as they were dosed.
- Tissue Collection: The urinary bladder and ureters were removed from the ventral and lateral sides of the uterus and vagina. The uterus and vagina were removed from the body, and excess fat and connective tissue were trimmed away. The vagina was removed from the uterus below the cervix, so that the cervix remained with the uterine body. The ends of the uterine horns were examined for the presence of any ovarian tissue. Gross observations of the uterus were recorded.
- Tissue Weights: The uterus was weighed to the nearest 0.1 mg. The uterus was then pierced and blotted to remove the luminal contents and weighed (blotted) to the nearest 0.1 mg. - Positive control:
- Rats were administered 17α-ethinyl estradiol as positive control for three consecutive days at a nominal dose level of 0.1 mg/kg.
- Details on results:
- IN-LIFE ANIMAL OBSERVATIONS
- Mortality/Moribundity
All animals survived to the scheduled humane euthanasia with no animals showing signs of moribundity.
- Clinical Observations
Animals administered the vehicle control or low dose of test material (125 mg/kg) exhibited no abnormal clinical signs. Rats administered 400 mg/kg exhibited greasy rectal area present in one rat on Day 2, two rats on Day 3, and three rats prior to euthanasia 24 hours following the last dose administration.
Administration of 17α-ethinyl estradiol did not elicit any abnormal clinical observations.
- Cage-side Observations
There were no cage-side post administration adverse observations during the study.
- Body Weight
Final body weight (88 % of control weight) and body weight gain of female rats administered 400 mg/kg test material were significantly decreased compared to the control group. Body weights of rats administered 125 mg/kg test material were not statistically changed.
NECROPSY PROCEDURES
- Uterine Weights
Administration of test material did not affect either wet or blotted uterine weights compared to controls. The positive control, 17α-ethinyl estradiol, resulted in significantly increased wet and blotted uterine weights compared to the vehicle control group.
- Performance Criteria
Vehicle control mean blotted uterine weights of animals were less than 0.04 % of body weight indicating the study met the performance criteria.
The assay was conducted using immature rats and administration of 17α-ethinyl estradiol via subcutaneous injection. The testing facility conducted this assay prior to the US EPA finalising and releasing the uterotrophic assay testing guideline (US EPA, 2009) and the stated preferences of the ovariectomised rat model. - Conclusions:
- Under the conditions of this study, oral administration of the test material at dose levels of 125 and 400 mg/kg (maximum tolerated dose) did not cause oestrogenic effects.
- Executive summary:
A study was conducted to evaluate the test material for its oestrogenic effects utilising the ovariectomised rat model in a Uterotrophic Assay conducted in accordance with the standardised guidelines OECD 440 and US EPA OPPTS 890.1600 under GLP conditions.
Ovariectomised Sprague-Dawley adult rats (8 per group) were orally administered 125 or 400 mg/kg of the test material, the vehicle control, or17α-ethinyl estradiol (positive control) for three consecutive days and then humanely euthanised. Dose levels were selected based on previous studies and dose levels above 400 mg/kg were found to cause mortality in adult female rats.
Body weights and clinical observations were performed daily. Approximately 24 hours following the final dose administration, the animals were humanely euthanised; the uteri were excised and wet and blotted uterine weights recorded.
All animals survived to the scheduled humane euthanasia with no animals showing signs of moribundity. Animals administered the vehicle control or low dose of test material exhibited no abnormal clinical signs. Rats administered 400 mg/kg exhibited greasy rectal area present in one rat on Day 2, two rats on Day 3, and three rats prior to euthanasia 24 hours following the last dose administration. Administration of 17α-ethinyl estradiol did not elicit any abnormal clinical observations.
Final body weight (88 % of control weight) and body weight gain of female rats administered 400 mg/kg test material were significantly decreased compared to the control group. Body weights of rats administered 125 mg/kg test material were not statistically changed.
Administration of test material did not affect either wet or blotted uterine weights compared to controls. The positive control, 17α-ethinyl estradiol, resulted in significantly increased wet and blotted uterine weights compared to the vehicle control group, indicating a positive response in the animal model.
Under the conditions of this study, oral administration of the test material at dose levels of 125 and 400 mg/kg (maximum tolerated dose) did not cause oestrogenic effects.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2011 to 26 October 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 456
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1550
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: steroidogenic pathway
- Species:
- human
- Strain:
- other: Human H295R cell line (adrenocarcinoma cell line)
- Details on test animals or test system and environmental conditions:
- CELL LINE
- Source
The cell line was obtained from the American Type Culture Collection (ATCC CLR-2128; Lot #7635054), Manassas, VA
- Stability of the cell line
The stability was monitored by the use of the following reference materials: forskolin and prochloraz. Two concentrations for each reference material were included on a QC plate each time the assay was performed and the fold change values for testosterone and estradiol were compared to the acceptable values from the guidelines. Additionally, basal production of testosterone and estradiol on the QC plate were compared to the acceptable values.
- Cell Culture and Plating Conditions
Cells were maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham with 15 mM HEPES, sodium bicarbonate, ITS+Premix and 2.5 % Nu-Serum (hormone concentrations in undiluted Nu- Serum: testosterone: 3754 pg/mL, estradiol: 3846 pg/mL) in a 5 % CO2 incubator at 37 ± 2 °C.
H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 μM 22R-hydroxycholesterol at the time of plating, dosing, and harvest. The concentration of 22R-hydroxycholesterol was chosen based on laboratory proficiency experiments previously conducted at the testing facility. The cells were plated into wells of a 24-well cell culture plate at a density of approximately 300 000 cells/mL. The cells were then placed into a 5 % CO2 incubator at 37 ± 2 °C for approximately 24 hours prior to chemical exposure. - Route of administration:
- other: Solutions added to culture plates
- Vehicle:
- DMSO
- Details on exposure:
- Test material was dissolved in DMSO to make a 200 mM stock and then serially diluted 1:10 in DMSO. 22R-hydroxycholesterol was dissolved in ethanol to make a 40 mM stock and then diluted in supplemented medium to a final concentration of 10 μM. Test material was then diluted 1:2000 in supplemented medium containing 10 μM 22R-hydroxycholesterol to prepare mastermix solutions. Forskolin and prochloraz were dissolved in DMSO to make 100 mM solutions and then serially diluted in DMSO. Forskolin and prochloraz were then diluted 1:2000 in supplemented medium containing 10 μM 22R-hydroxycholesterol.
The final concentration of DMSO in the medium was held constant at 0.05 % (v/v). The final ethanol concentration in the supplemented medium was 0.025 % (v/v). - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Plates were incubated for 48 ± 2 hours
- Frequency of treatment:
- Once
- Dose / conc.:
- 1 other: µM
- Remarks:
- forskolin
- Dose / conc.:
- 10 other: µM
- Remarks:
- forskolin
- Dose / conc.:
- 0.1 other: µM
- Remarks:
- prochloraz
- Dose / conc.:
- 1 other: µM
- Remarks:
- prochloraz
- Dose / conc.:
- 0 other: µM
- Remarks:
- test material
- Dose / conc.:
- 0.001 other: µM
- Remarks:
- test material
- Dose / conc.:
- 0.01 other: µM
- Remarks:
- test material
- Dose / conc.:
- 0.1 other: µM
- Remarks:
- test material
- Dose / conc.:
- 1 other: µM
- Remarks:
- test material
- Dose / conc.:
- 10 other: µM
- Remarks:
- test material
- Dose / conc.:
- 100 other: µM
- Remarks:
- test material
- No. of animals per sex per dose:
- All concentrations were tested in replicates of 3/plate; 3 independent runs were performed.
- Control animals:
- other: Reference materials and solvent controls were used
- Details on study design:
- CHEMICAL EXPOSURE
Prior to dosing, the cells were checked microscopically for attachment and proper morphology. The medium was removed from the cells and replaced with 1 mL of medium containing 10 μM 22R-hydroxycholesterol and the required concentration of test or control material.
A concurrent QC plate was included with each of the independent runs of the test material plates. The QC plate was prepared and exposed to the reference materials in the same manner.
After adding the dosing solutions, the plates were incubated in a 5 % CO2 incubator at 37 ± 2 °C for 48 ± 2 hours. After the 48 ± 2 hour exposure, each well was examined under the microscope and images were taken of the vehicle control wells in addition to the two greatest non-cytotoxic concentrations (based on observation under the microscope). The media was collected from all wells in two equal aliquots and stored at -80 ± 10 °C until shipment to the analytical laboratory for hormone measurement. After media removal, cell viability was measured in all plates by MTT assay.
CYTOTOXICITY ASSAY
Cell viability was monitored by MTT assay after the 48 ± 2 hour exposure. On the QC plates, wells designated to receive methanol (control wells for cell death measurements) were rinsed twice with PBS, then incubated in methanol for 30 minutes at room temperature. After the methanol incubation, the methanol-treated wells were rinsed again with PBS three times. Following media removal and/or methanol treatment, 0.5 mL of 0.5 mg/mL MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol was added to each well of the test material and QC plates. The plates were incubated at 37 ± 2 °C in a 5 % CO2 incubator for 3 hours. Following the 3 hour incubation, the MTT solution was removed and 0.5 mL of isopropanol was added to each well. Plates were incubated at room temperature for 20 minutes with shaking. Following this incubation, absorbance at 570 and 650 nm were measured on a Packard Fusion plate reader. The absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT value for each well.
The change in cell viability was determined by comparing treated wells to the solvent control wells. A greater than 20 % reduction in cell viability was considered evidence of cytotoxicity.
PRECIPITATION ASSAY
Final dilutions of test material in supplemented media were observed visually for evidence of precipitation. If precipitation was observed, the concentration was considered insoluble and was excluded from further analysis.
HORMONE MEASUREMENT SYSTEM
Testosterone and estradiol levels were measured using HPLC/MS-MS. The method detection limit is 100 pg/mL for testosterone and 10 pg/mL for estradiol. The resulting minimum basal production levels are 500 pg/mL for testosterone and 25 pg/mL for estradiol. - Details on results:
- DOSES ANALYSED
The suitable top concentration that could be analysed was 0.1 μM in Runs 2 and 4 because of cytotoxicity greater than 20 % at higher concentrations. Contamination was observed at the 1 μM concentration in Run 4. This concentration was not included in the statistical analysis because of cytotoxicity greater than 20 %. As a result, the contamination is not expected to have an impact on the integrity of the data. In Run 3, the suitable top concentration that could be analysed was 1 μM because of cytotoxicity greater than 20 % at higher concentrations. No solubility issues were noted at any of the concentrations tested in the assay.
ASSAY RESULTS
In three independent runs of the assay, a statistically significant reduction in testosterone was observed at the 0.001 μM concentration in Run 4; however, this was not dose dependent.
Statistically significant induction or inhibition was not observed at any other concentrations or at any concentration in Runs 2 or 3 for testosterone. Statistically significant changes in estradiol were not observed at any of the concentrations that could be analysed in any of the three independent runs of the assay. Contamination was observed at one concentration (1 μM) in Run 4. This concentration was not included the statistical analysis.
ASSAY ACCEPTANCE CRITERIA
In all three independent runs of the assay, the basal production of testosterone and estradiol on the quality control plates was above the required levels. In addition, the fold change required after induction with 10 μM forskolin and inhibition with 1 μM prochloraz on the quality control plates met the requirements for both the testosterone and estradiol analyses.
The coefficients of variation for solvent control replicate wells for testosterone within a plate based on absolute concentrations were 1.51 % for Run 2, 8.09 % for Run 3, and 4.55 % for Run 4. The coefficients of variation for solvent control replicate wells for estradiol within a plate based on absolute concentrations were 5.02 % for Run 2, 10.5 % for Run 3, and 0.85 % for Run 4. The between plate coefficient of variation for solvent controls based on fold change was 4.70 % for testosterone and 5.82 % for estradiol. - Conclusions:
- The test material showed statistically significant inhibition of testosterone production at one concentration (0.001 µM) in one run. The induction was not dose dependent.
- Executive summary:
A study was conducted to evaluate the ability of test material to affect the steroidogenic pathway beginning with the sequence of reactions occurring after the gonadotropin hormone receptors through the production of testosterone and estradiol/estrone. The assay used the H295R human adrenocarcinoma cell line and was conducted in accordance with the standardised guidelines OECD 456 and US EPA OPPTS 890.1550 under GLP conditions.
The test material was tested in the assay in DMSO at the following concentrations: 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM. The stability of the cell line was monitored by the use of forskolin and prochloraz as reference materials.
The test and reference materials, along with solvent controls, were tested in replicates of 3/plate, with the exception of the solvent controls on the quality control (QC) plate. Six solvent control wells were analysed on the QC plate. The H295R supplemented medium used in the assay at the time of plating, dosing, and harvest contained 10 μM 22R-hydroxycholesterol. The duration of exposure was 48 ± 2 hours. A QC plate containing two doses of the reference materials forskolin and prochloraz was run each time the assay was performed. Cell viability was assessed after the 48 ± 2 hour exposure using the MTT assay. Testosterone and estradiol levels were measured using HPLC/MS-MS.
The suitable highest concentration of test material that could be analysed in the steroidogenesis assay was 0.1 μM in Runs 2 and 4 and 1 μM in Run 3 based on cytotoxicity results. No solubility issues were noted at any of the concentrations tested.
In three independent runs of the steroidogenesis assay, statistically significant inhibition of testosterone was observed at one concentration of test material (0.001 μM) in one run.
Statistically significant induction or inhibition was not observed at any other concentrations or in the other two independent runs of the assay. Statistically significant induction or inhibition of estradiol was not observed.
In conclusion, the test material showed statistically significant inhibition of testosterone production at one concentration (0.001 µM) in one run. The induction was not dose dependent.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2011 to 26 October 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1200
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: Human recombinant aromatase inhibition
- Species:
- human
- Strain:
- other: Human recombinant microsomes
- Details on test animals or test system and environmental conditions:
- - Human Recombinant Microsomes
Human recombinant microsomes were purchased from Gentest™. The product name was Human CYP19 (Aromatase) and P450 reductase Supersomes™. The package insert (batch data sheet) provided values for protein concentration, cytochrome c reductase activity, and aromatase activity. The microsomes were stored at -80 ± 10 °C.
- Human Recombinant Microsome Preparation
Initial preparation of the human recombinant microsomes involved thawing the microsomes rapidly in a 37 ± 2 °C water bath and performing a two-step dilution. Following thawing, microsomes (e.g., 6.9 and 7.4 mg/mL) were placed in an ice bath and diluted to 0.8 mg/mL with buffer (0.1 M sodium phosphate, pH 7.4). Microsomes were further diluted to 0.008 mg/mL and aliquoted into individual vials. After aliquoting the microsomes into individual vials, the vials that were not planned for immediate use were returned to the -80 ± 10 °C freezer for storage (information regarding stability to freeze thaw cycles was provided on the batch data sheet). This minimised freeze-thaw cycles to no more than one. The assay used vials containing 0.008 mg/mL protein and final concentration was approximately 0.004 mg/mL of microsomal protein per assay tube. - Route of administration:
- other: Test material incorporated into test vessels
- Vehicle:
- DMSO
- Details on exposure:
- The test material was formulated in dimethylsulfoxide (DMSO). The total volume of DMSO used in each assay was 1 % of the total assay volume in order to minimise the potential of this vehicle to inhibit the aromatase (CYP19). Fresh dilutions of the stock solution of the test material were prepared on the day of use such that the target concentration of the test material was achieved by the addition of 20 μL of the dilution to a 2 mL total assay volume.
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- Tubes were incubated for approximately 15 minutes
- Frequency of treatment:
- Once
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0.001 other: M
- Dose / conc.:
- 0.01 other: M
- Remarks:
- Additional concentrations:10^-4.5 and 10^-3.5 M
- No. of animals per sex per dose:
- All concentrations were tested in triplicate; 3 independent runs were performed.
- Control animals:
- other: Vehicle control, full activity control and background activity control took place
- Details on study design:
- SUBSTRATE
The substrate for the aromatase assay was androstenedione (4-Androstene-3,17-dione or ASDN). Non-radiolabelled and radiolabelled androstenedione with a specific activity of 26.3 Ci/mmol ([1β-³H]-androstenedione, [³H] ASDN) were used.
OTHER ASSAY COMPONENTS
- Buffer: The assay buffer was 0.1 M sodium phosphate buffer, pH 7.4. Sodium phosphate monobasic and sodium phosphate dibasic were used to prepare this buffer. 0.1 M solutions of each reagent were prepared in purified water and then combined to achieve a final pH of 7.4.
- Propylene Glycol: Propylene glycol was added to the assay directly.
- NADPH: NADPH (β-nicotinamide adenine dinucleotide phosphate, reduced form, tetrasodium salt) was the required co-factor for CYP19. A 6 mM stock solution was prepared in assay buffer and the final concentration in the assay was 0.3 mM NADPH. NADPH was prepared fresh each day the assay was performed and was kept on ice prior to use in the assay.
TEST SYSTEM
Recombinant microsomes (Human CYP19 + P450 Reductase Supersomes™) were used as the test system for this study.
PRELIMINARY EXPERIMENTS TO DETERMINE SUITABILITY OF MICROSOME PREPARATIONS
- Protein Assay: Protein content of the microsomes was 6.9 and 7.4 mg/mL.
- Cytochrome P450 (CYP19) Aromatase Activity: Aromatase activity of the microsome preparation was 2.8 and 6.0 pmol product/(min x pmol P450).
AROMATASE ASSAY METHOD
The assays were performed in 13 x 100 mm test tubes maintained at 37 ± 2 °C in a shaking water bath. Propylene glycol, [³H] ASDN, NADPH and assay buffer were combined in the test tubes, with or without the test material for a total volume of 1 mL. The final concentrations of the major components were microsomal protein 0.004 mg/mL, NADPH 0.3 mM, [³H]ASDN 100 nM and propylene glycol 5 %.
The test tubes and microsomal suspensions were placed at 37 ± 2 °C in the water bath for approximately 5 minutes prior to the initiation of the assay by the addition of 1 mL of the diluted microsomal suspension. The total assay volume was 2 mL. The tubes were then incubated for approximately 15 minutes at 37 ± 2 °C. The reactions were then terminated by the addition of 2 mL of ice-cold methylene chloride and vortexed-mixed for approximately 5 seconds and placed on ice. The tubes were then re-vortex mixed for an additional 20 to 25 seconds to extract the unreacted ASDN. Tubes were centrifuged for 10 min at 200 x g (37 ± 2 °C). The methylene chloride layer was removed (bottom layer) and discarded and the aqueous layer was extracted two more times. Two 0.5 mL aliquots of the top aqueous layers were then transferred to duplicate liquid scintillation vials containing 10 mL of liquid scintillation cocktail (Opti-Fluor 60131989) and then mixed.
Analysis of the samples was performed using (Packard TriCarb LSC 2910TR, serial DG03117657). Radioactivity found in the aqueous fractions is from the ³H2O formed upon hydrolysis of [³H] ASDN. One H2O molecule is released per molecule of ASDN converted to oestrogen in a stereospecific reaction. Therefore the amount of oestrogen product formed was determined by dividing the total amount of ³H2O formed by the specific activity of the [³H] ASDN substrate (expressed in dpm/mL). Results are presented as the activity of the enzyme reaction and expressed in nmol (mg protein)^-1 min^-1.
The test material was tested in three independent runs and for each run each concentration was performed in triplicate (N = 3). In each run, full enzyme activity (Full Activity Control) and background activity (Background Activity Control) samples were included.
Full activity control samples contain substrate [³H] ASDN, NADPH, propylene glycol, buffer, test material in DMSO, and microsomes. Background activity controls contained the same components as the full enzyme activity control samples except NADPH was excluded. Four full activity controls and four background activity controls were included with each run of the assay. All controls were split in half so that two tubes (for full and background activity) were run at the beginning of the assay and two of each (full and background activity) were run at the end of each assay.
DETERMINATION OF THE RESPONSE OF AROMATASE ACTIVITY TO THE TEST MATERIAL
Each independent run tested the response of aromatase activity in the presence of eight concentrations of the test material run in triplicate. Three types of control samples were included for each run. These included full enzyme (aromatase) activity controls, background activity controls and positive control.
After completion of the first run, the data were reviewed. Concentration of the test material used in the latter runs was not adjusted based upon the results of the first run.
SOLUBILITY ASSESSMENT OF TEST MATERIAL
Solubility of the test material was assessed in the first run of the assay by visual observation.
- Solubility Assessment and Concentration Ranges
If insolubility (cloudiness or a precipitate) was visually observed at the highest concentration
(10^-3 M), then the highest concentration would be adjusted for the second and third runs at the highest concentration that appeared soluble using log or half-log concentrations; i.e., 10^-3.5, 10^-4 M, etc. Concentrations lower than 10^-5 M for the highest concentration were not tested.
The lowest concentration to be tested was 10^-10 M, but lower concentrations were required to obtain the “top of the curve”. That is, the full enzymatic activity was obtained at the two lowest concentrations of the test material in order to define the top of the concentration-response curve. - Positive control:
- The known aromatase inhibitor, 4-hydroxyandrostendione (4OH-ASDN; Formestane), was used as the positive control for aromatase inhibition. The 4OH-ASDN was formulated in 100 % dimethylsulfoxide (DMSO). Fresh dilutions of the stock solution were prepared on the day of use. Dilutions were prepared such that the target concentrations of control material could be achieved by the addition of 20 μL of the dilution to a 2 mL total assay volume with final DMSO concentrations ≤1 %.
POSITIVE CONTROL ASSAY
The minimum level of mean aromatase activity in the full activity control samples was 0.180 nmol/mg-protein/min, well above the 0.100 nmol/mg-protein/min minimum acceptable activity limit set forth in the guideline. The mean background control activity was ≤15 % of the full activity control, the limit established in the guideline. The concentration response curve data generated for 4OH-ASDN were compared to the performance criteria conditions. - Details on results:
- AROMATASE ASSAY RESULTS
The results of the test material analyses are presented in Tables 3 to 5. The suitable top concentration for use in the aromatase assays was 10^-4 M, based on test material solubility. The positive control inhibitor 4OH-ASDN was included with each run each time the aromatase assay was performed to ensure results passed the performance criteria. In three independent runs of the aromatase assay, test material aromatase activity was determined to be 96.9 % of control.
In three independent runs of the assay, the test material tested at concentrations of 10^-10 to 10^-4 M resulted in little decrease in aromatase activity relative to full activity controls. Aromatase activity in the presence of 10^-4 M was determined to be 96.9 % of control activity. Based on the data interpretation criteria established for the assay, the test material was classified as a non-inhibitor of the aromatase enzyme.
AROMATASE ASSAY ACCEPTANCE CRITERIA
In three independent runs of the positive control assay (4OH-ASDN), the mean Hill slope, IC50, bottom curve (%), and top curve (%) were all within the acceptable ranges.
All independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be definitive.
QUALITY CONTROL ANALYSIS ACCEPTANCE CRITERIA
In three independent runs of the assay, all runs were within specifications. In addition, the mean background activity controls were ≤15 % of the full activity controls. - Conclusions:
- The test material was soluble across the exposure range tested (10^-10 to 10^-4 M) and was determined to be a non-inhibitor for aromatase activity.
- Executive summary:
A study was conducted to evaluate the ability of the test material to act as an inhibitor of aromatase activity using human CYP19 (aromatase) and P450 reductase supersomes in accordance with the standardised guideline US EPA OPPTS 890.1200 under GLP conditions.
The substrate for the assay is androstenedione (ASDN), which is converted by aromatase to estrone. The final concentrations of the test material tested in the Aromatase assay were 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4.5 and 10^-4 M.
Four independent runs of the aromatase assay were conducted; however, the first run was considered to be invalid due to ASDN, and not 4OH-ASDN, being used as the positive control. As such, the first run was excluded from the analysis. In each independent run, each concentration of the test material was tested in replicates of three. In addition, the positive control inhibitor 4-hydroxyandrostenedione (4OH-ASDN) was included each time the aromatase assay was performed. Increasing concentrations of 4OH-ASDN decrease the aromatase activity in a concentration dependent manner.
In three independent runs of the assay, increasing concentrations of the test material showed little decrease in aromatase activity. Visual inspection during the first and second test runs revealed precipitation at 10^-3 and 10^-3.5 M. The test material was found to be soluble in the assay buffer at concentrations of ≤10^-4 M. Thus, the suitable top concentration for use in the aromatase assay was established at 10^-4 M.
The mean of the activity level at the highest soluble test concentration can be used to determine whether the test material is an inhibitor, non-inhibitor, or equivocal. The test material produced a mean aromatase activity level of 96.9 % of the control activity ± 2.7 % SD at the highest soluble test concentration of 10^-4 M and was therefore considered to be a non-inhibitor.
The test material was soluble across the exposure range tested (10^-10 to 10^-4 M) and was determined to be a non-inhibitor for aromatase activity.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2011 to 25 October 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1150
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: Androgen receptor binding
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age: 90 days
- Days after castration: < 1
RAT PROSTATE CYTOSOL PREPARATION AND INFORMATION
- Protein Concentration: 6.2 mg/mL
- Method of Determination: Bradford Method
- Supplier and Product: Bio-Rad Dye Reagent Concentrate
- Catalogue Number: 500-0006
- Batch/Lot Number: 210007463
Isolation Procedure
- Ensure the homogeniser probe is pre-chilled before use, by placing it in a beaker of low-salt TEDG buffer on ice.
- Inspect prostate tissue for healthy appearance (no fibrous, inflamed, oedematous or infected appearance) and discard any tissues that appear compromised; trim excess fascia if necessary.
- Weigh prostate tissues or use recorded tissue weights; calculate total weight and add to a beaker of low-salt TEDG buffer in ice bath, at 10 mL of buffer/g tissue.
- Mince with a scalpel blade and fine scissors until all pieces are small 1-2 mm cubes.
- Homogenise tissues at 4 °C with a pre-chilled Polytron homogeniser. For a Polytron PT2100, use setting 3, and 3 short 4 second bursts of power spaced at 20 second intervals and place probe into ice cold TEDG buffer to cool it down between bursts.
- Transfer homogenates to pre-cooled centrifuge tubes, balance, and centrifuge at 30 000 x g for 30 minutes in a centrifuge cooled to 4 °C. For centrifugation using a Sorvall RC5B centrifuge, use an SS-34 rotor and set centrifuge speed to 16 000 RPM.
- The resulting supernatant contains the low-salt cytosolic receptor. Pool the supernatant from all rats.
- Aliquot into labelled tubes at a volume determined to reduce waste, as cytosolic supernatant cannot be refrozen once thawed (aliquoting multiple volumes allows flexibility). Discard after 6 months unless revalidation run is performed.
- Determine protein content for each batch of cytosol using a method that is compatible with buffers that contain DTT. Typical protein values are 1 to 4 mg/mL. - Route of administration:
- other: in vitro
- Vehicle:
- DMSO
- Details on exposure:
- PREPARATION OF STOCK SOLUTIONS
A 200 mM EDTA stock solution was prepared and stored at 4 ± 2 °C. A 1 M sodium molybdate solution was also prepared along with a 1 M Tris buffer (pH adjusted to 7.4). These solutions were then used to prepare Low-salt TEDG Buffer (10 mM Tris, 1 mM sodium molybdate, 1.5 mM EDTA, 10 % glycerol and 1 mM DTT (added immediately before use), pH 7.4 (cooled to 4 ± 2 °C before adjusting to pH 7.4 and stored at 4 ± 2 °C up to 3 months)).
A 600 μM stock solution of triamcinolone acetonide was prepared in 100 % ethanol and diluted/aliquoted into 60 μM solutions and stored at -20 ± 2 °C.
The 60 % hydroxyapatite (HAP) slurry was prepared one day before use. The HAP was gently mixed with 50 mM buffer in a graduated cylinder, and refrigerated for approximately 2 hours at 4 ± 2 °C. The HAP was then washed twice as follows. The supernatant was removed and the HAP was re-suspended again in 50 mM Tris buffer (4 ± 2 °C). The slurry was mixed gently and allowed to settle for approximately 2 hours at 4 ± 2 °C. After the second wash, the HAP slurry settled overnight (at least 8 to 10 hours at 4 ± 2 °C).
The next day (day of use), the volume of HAP on the graduated cylinder was noted. The supernatant was removed and the HAP was re-suspended to a final volume of 60 % HAP and 40 % cold 50 mM Tris buffer. The HAP slurry was well-suspended and ice-cold when used in the separation procedure.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material solutions with a concentration of up to 10^-4 M (the limit concentration for the assay) can be prepared.
- Concentration in vehicle: The final concentration of DMSO in the assay medium was limited to ~3.2 % (v/v). - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- The tubes were incubated for 16 to 20 hours
- Frequency of treatment:
- Once
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0.001 other: M
- Dose / conc.:
- 0.01 other: M
- No. of animals per sex per dose:
- All concentrations were tested in triplicate; 3 independent runs were performed.
- Control animals:
- other: Yes; concurrent solvent, positive and weak positive controls were run
- Details on study design:
- SOLUBILITY/PRECIPITATION ASSAY
The limit of test material solubility was determined by visual observation. Compound solubility was determined in 100 % DMSO. In addition, the solutions were examined closely after the overnight incubation (as the test material may precipitate in the assay tube mixtures).
ASSAYS
WORKING ASSAY BUFFER PREPARATION
Summary of Assay Conditions:
- Source of receptor: Rat prostate cytosol
- Concentration of radioligand: 1 nM
- Concentration of receptor: Sufficient to bind 10 - 15 % of radioligand
- Temperature: 4 ± 2 °C
- Composition of assay buffer: Tris 10 mM (pH 7.4); EDTA 1.5 mM; Glycerol 10 % (v/v); Protease Inhibitor 0.5 % (v/v); DTT 1 mM and Sodium Molybdate 1 mM.
On the day of assay, the Working Assay Buffer, or TEDG+PI buffer (10 mM Tris, 1 mM sodium molybdate, 1.5 mM EDTA, 10 % glycerol and 1 mM DTT, 0.5 % Protease Inhibitor (v/v), pH 7.4) was prepared using the TEDG buffer.
[³H]-R1881 PREPARATION
[³H]-R1881 was prepared on the day of assay. The specific activity was adjusted for decay over time prior to performing dilutions. The specific activity was calculated on the day of the assay using the following equation:
SAadjusted (Fraction Isotope Remaining) = SA * e^(-Kdecay*Time)
Where:
SA = the specific activity on the packaging date
Kdecay = the decay constant for tritium (equal to 1.54 x 10^-4/day).
Time = days since the date on the stock bottle from the manufacturer.
The [³H]-R1881 was diluted with TEDG + PI buffer so that each assay tube contained 1 nM final concentration of [³H]-R1881 using the following procedure:
The specific activity was converted from Ci/mmole to nM. If SA = X Ci/mmole, and Y = concentration of radiolabel, then X Ci/mmole was converted to nM and the SA activity adjusted for decay over time by the following conversion:
(Y mCi/mL / X Ci/mmole) * 1 Ci/1000 mCi * 10^6 nmole/mmole * 1000 mL/L = (Y/X) * 10^6 nM
A 10 nM diluted stock of the [³H]-R1881 was prepared so that 30 μL in a total volume of 300 μL per assay tube will give a final concentration of 1 nM. The 30 nM [³H]-R1881 was kept on ice.
ASSAY PREPARATIONS
12 x 75 mm siliconised tubes were used for the assay. 30 µL of 10 nM [³H]-R1881 (1 x 10^-8 M) and 50 μL triamcinolone acetonide (60 μM working solution) were added to all tubes. For the 3 tubes at the beginning of assay and at the end of assay, 100X inert R1881 (30 μL of 1 μM) was also added. These are the nonspecific binding tubes. The tubes were placed in a speed-vac and dried. An aliquot of cytosol was thawed on ice and diluted to the predetermined optimal protein concentration.
INDIVIDUAL TUBES
For the assay tubes, 10 μL of each concentration of test material and control was added, followed by 300 μL of the diluted cytosol. The temperature of the tubes and contents were kept at 4 ± 2 °C prior to the addition of the cytosol. The assay tubes were vortexed after additions and incubated at 4 ± 2 °C for 16 to 20 hours on a rotator.
SEPARATION OF BOUND [³H]-R1881 FROM FREE [³H]-R1881
The AR assay tubes were removed from the rotator and placed in an ice-water bath. A repeating pipette was used to add approximately 500 μL of ice cold HAP slurry (60 % in 50 mM Tris buffer) to fresh new 12 x 75 mm siliconised assay tubes. 100 μL of each incubation tube was transferred to the appropriate labelled tubes containing the HAP. The tubes were vortexed for approximately 10 seconds at approximately 5 minute intervals for a total of approximately 20 minutes with tube remaining in the ice-water bath between vortexing.
Following the vortexing step, approximately 2 mL of the cold 50 mM Tris buffer was added, quickly vortexed, and centrifuged at 4 ± 2 °C for approximately 10 minutes at 700 x g. After centrifugation, the supernatant containing the free [³H]-R1881 was immediately decanted and discarded. The HAP pellet contained the androgen receptor bound [³H]-R1881.
Approximately 2 mL of ice-cold 50 mM Tris buffer was added to each tube and vortexed to re-suspend the pellet. The tubes were centrifuged again at 4 ± 2 °C for approximately 10 minutes at approximately 700 x g. The supernatant was quickly decanted and discarded. The wash and centrifugation steps were repeated three more times. After the final wash, the supernatant was decanted. The assay tubes were allowed to drain briefly for approximately 30 seconds.
EXTRACTION AND QUANTIFICATION OF [³H]-R1881 BOUND TO AR
Approximately 2 mL of absolute ethanol was added to each assay tube. The tubes were allowed to sit at room temperature for approximately 15 to 20 minutes, vortexing for approximately 10 seconds at approximately 5-minute intervals. The assay tubes were centrifuged for approximately 10 minutes at approximately 700 x g. The supernatant was decanted into a 20 mL scintillation vial containing approximately 14 mL scintillation cocktail (Perkin Elmer Opti-Fluor). The vial was capped and shaken. The vials were placed in a scintillation counter (Perkin Elmer Tri-Carb 2910TR Liquid Scintillation Analyzer Model B2910) and each vial was counted for at least one minute with quench correction for determination of DPMs per vial.
Standards (³H, 14C and background) were used to verify accurate counting, and the liquid scintillation analyzer has an enhanced Instrument Performance Assessment (IPA) for monitoring efficiencies, backgrounds, E2/B and Chi-square values for ³H and 14C over the life of the instrument. - Positive control:
- POSITIVE AND WEAK POSITIVE REFERENCE CONTROLS
The positive control, R1881, strongly binds ARs and was included to ensure that the run was properly performed and to allow an assessment of variability in the conduct of the assay across time. Final concentrations of unlabelled R1881 ranged from 1 x 10^-6 to 1 x 10^-11 M. Fresh 10 mM R1881 stock was diluted 1:10 (1 mM R1881 solution) and then serial dilutions of the reference standard were performed in DMSO (final concentration of approximately 3.2 %).
The weak positive control was dexamethasone. A 30 mM stock was prepared in DMSO and serially diluted. The concentration range tested for the weak positive control was from 1 x 10^-3 to 1 x 10^-10 M with DMSO kept at approximately 3.2 %. - Details on results:
- CONCENTRATION RANGE FOR THE TEST MATERIAL
The final concentrations of the test material to assess precipitation were 10^-4 and 10^-3 M. The suitable top concentration for use in the first and third valid independent runs was 10^-4 M as precipitation was observed with 10^-3 M. The suitable top concentration for use in the second valid independent run was 10^-5 M as precipitation was observed with 10^-4 M.
The final concentrations of the test material assessed in the binding assays were: 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 M for the first valid independent run and 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 M for the second and third valid independent runs.
RESULTS
All three runs resulted in very similar results indicating the assay was functioning properly. In the first valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-binding” for this run. Given that the highest concentration of dexamethasone was not prepared, the bottom plateau level is not reached. Historical data suggests it is typically around 0 %. In order to generate a LogIC50, the bottom plateau was constrained to 0 % and the resultant LogIC50 of dexamethasone was -4.4 M. The LogIC50 of R1881 was -9.9 M. Also, the DPM of the third replicate of 10^-4 M test material was ~ 3x higher than the mean total binding DPM. This suggests that at least a portion of the HAP pellet was accidentally decanted into the scintillation vial. This data point was removed from the analysis.
In the second valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-binding” for this run. The weak positive control dexamethasone had a LogIC50 of -4.4 M while the LogIC50 of R1881 was -9.0 M.
Finally, in the third valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-interacting” for this run. The weak positive control dexamethasone had a LogIC50 of -4.6 M while the LogIC50 of R1881 was -9.0 M.
The mean relative binding affinity, or RBA (calculated by dividing the LogIC50 of the control/test material by the LogIC50 of the positive control R1881) was 0.5 for dexamethasone. The RBA could not be calculated for the test material.
The test material was classified as a “non-binder” in all three valid independent runs and thus has a final classification of “non-binder.”
BINDING ASSAY ACCEPTANCE CRITERIA
In the first valid independent run, R1881 and dexamethasone were prepared at concentrations that were slightly different than the test guideline. R1881 was prepared at -7.48, -8.48, -9.48, -10.48, -11.48 and -12.48 Log[M] instead of -6, -7, -8, -9, -10 and -11 Log[M]. The curve for R1881 fit the acceptance criteria and performed within the testing facility historical data. Dexamethasone was prepared at -3, -4, -5, -6, -7, -8, -9 and -10 Log[M] instead of -4, -5, -6, -7, -8, -9, -10 and -11 Log[M]. The resulting curve for dexamethasone fit two of the three criteria (Hill Slop and Top Plateau) and the LogIC50 for dexamethasone performed within the testing facility historical data, indicating that despite the differences in concentrations the assay was functioning properly and the results were accurate.
In all three valid independent runs of the assay, increasing concentrations of unlabelled R1881 displaced [³H]-R1881 from the receptor in a manner consistent with one-site competitive binding, and the ligand depletion was held below 15 %. Also, the solvent did not alter the assay sensitivity or reliability.
Finally, the data were within the acceptable ranges specified with the following exception: In the first valid independent run of the assay, the bottom plateau level for dexamethasone was not able to be accurately calculated (bottom plateau level = N/A; compared to the specified range of -12 to 12 %). This is because of the deviations in the concentration range.
These deviations were minor and not considered to reflect true deviation from the suggested ranges outlined in the guideline. Therefore, all three independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be valid.
No data were excluded from either evaluation or interpretation due to excessive precipitation with addition of test material in any independent run of the assay. - Conclusions:
- The test material was classified as a “non-binder” in all three valid independent runs and thus has a final classification of “non-binder.”
- Executive summary:
A study was conducted to evaluate the ability of the test material to interact with the androgen receptors (ARs) isolated from rat prostates in accordance with the standardised guideline US EPA OPPTS 890.1150 under GLP conditions.
Preliminary assessments of precipitation were conducted in order to identify a suitable top concentration of the test material for use in the binding assays. The concentrations assessed were 10^-4 and 10^-3 M.
The final concentrations of the test material assessed in the binding assays were: 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 M for the first valid independent run and 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 M for the second and third valid independent runs.
All concentrations were tested in replicates of 3. In addition, DMSO solvent control tubes (6 replicates) were prepared to assess total binding. These replicates included the radioligand, cytosol (containing the ARs) and solvent but without the competitor R1881. The total binding tubes allowed for the identification of maximal binding of [³H]-R1881. Non-specific binding (NSB) was also assessed in replicates of 6 by determining the [³H]-R1881 bound in the presence of 100-fold excess unlabelled R1881. Data was NSB subtracted, normalised to total binding and presented as % specific binding. Finally, 30 μL of [³H]-R1881 was added to scintillation vials (n = 6) in order to determine both total radioligand added and to calculate the percentage of total radioligand added to the tube that is bound to ARs. The duration of incubation at approximately 4 °C was 16 to 20 hours. A complete concentration response curve for the positive control R1881 and the weak positive control dexamethasone was run each time the binding assay was performed.
In the first valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-binding” for this run. The weak positive control dexamethasone had a LogIC50 of -4.4 M while the LogIC50 of R1881 was -9.9 M.
In the second valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-binding” for this run. The weak positive control dexamethasone had a LogIC50 of -4.4 M while the LogIC50 of R1881 was -9.0 M.
Finally, in the third valid independent run, the mean specific binding was > 75 % at every soluble concentration tested, classifying it as “non-interacting” for this run. The weak positive control dexamethasone had a LogIC50 of -4.6 M while the LogIC50 of R1881 was -9.0 M.
The mean relative binding affinity, or RBA (calculated by dividing the LogIC50 of the control/test material by the LogIC50 of the positive control R1881) was 0.5 for dexamethasone. As the test material was classified as “non-interacting” for all three runs, the RBA could not be calculated.
Under the conditions of this study, the test material was classified as a “non-binder” in all three valid independent runs and thus has a final classification of “non-binder.”
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2011 to 25 October 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1250
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: Estrogen receptor binding
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age: 12 to 13 weeks
- Days after ovariectomy: 7 days
RAT UTERINE CYTOSOL PREPARATION AND INFORMATION
- Protein Concentration: 1.10 mg/mL
- Method of Determination: Bradford Method
- Supplier and Product: Bio-Rad Dye Reagent Concentrate
- Catalogue Number: 500-0006
- Batch/Lot Number: 210007463
Isolation Procedure
- Inspect uterine tissue for signs of residual ovarian tissue after ovariectomy (e.g., uterine imbibition) and discard tissue that was compromised.
- Weigh trimmed uterus, if weights not provided, and place in ice-cold TEDG buffer + PI at a ratio of 0.1 g of tissue per 1.0 ml TEDG + PI buffer. Homogenise the tissue using a Polytron (PT 35/10) or similar homogeniser for 3 to 5 bursts (~5 seconds per burst).
- Transfer homogenate to pre-cooled centrifuge tubes and centrifuge for 10 minutes at 2500 x g (Sorval RC SS34 rotor at 4500 RPM) at 4 °C. The supernatant contains the ER.
- Transfer the supernatant to pre-cooled ultracentrifuge tubes and centrifuge at 105 000 x g (Beckman 50.2TI rotor at 34,000 RPM) for 60 minutes at 4 °C. Discard the pellet.
- Keeping cytosol ice-cold, pool the cytosol supernatants containing ER.
- Determine protein content for each batch of cytosol using a method that is compatible with buffers that contain DTT. Typical protein values are 1 to 4 mg/mL.
- Aliquoted cytosol (1 to 6 mL aliquots) either for immediate use in ER binding assay or for storage at -80 °C. - Route of administration:
- other: in vitro
- Vehicle:
- DMSO
- Details on exposure:
- PREPARATION OF STOCK SOLUTIONS
A 200 mM EDTA stock solution was prepared and stored at 4 ± 2 °C. A 1 M Tris buffer was prepared and the pH adjusted to 7.4. These solutions were then used to prepare 2x TEG Buffer (20 mM Tris, 3 mM EDTA, 20 % glycerol, pH 7.4 (cooled to 4 ± 2 °C before adjusting to pH 7.4 and stored at 4 ± 2 °C up to 3 months)).
The 60 % hydroxyapatite (HAP) slurry was prepared one day before use. The HAP was gently mixed with ~3x volume of TEDG + PI buffer in a graduated cylinder, and refrigeratedfor approximately 2 hours at 4 ± 2 °C. The HAP was then washed twice as follows. The supernatant was removed and the HAP was re-suspended again in ~3x fresh TEDG + PI buffer (4 ± 2 °C). The slurry was mixed gently and allowed to settle for approximately 2 hours at 4 ± 2 °C. After the second wash, the HAP slurry settled overnight (at least 8 to 10 hours at 4 ± 2 °C).
The next day (day of use), the volume of HAP on the graduated cylinder was noted. The supernatant was removed and the HAP was re-suspended to a final volume of 60 % HAP and 40 % cold TEDG + PI. The HAP slurry was well-suspended and ice-cold when used in the separation procedure.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material solutions with a concentration of up to 10^-4 M (the limit concentration for the assay) can be prepared.
- Concentration in vehicle: The final concentration of DMSO in the assay medium was limited to 2 % (v/v). - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- The tubes were incubated for 16 to 20 hours
- Frequency of treatment:
- Once
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0.001 other: M
- Dose / conc.:
- 0.01 other: M
- No. of animals per sex per dose:
- All concentrations were tested in triplicate; 3 independent runs were performed.
- Control animals:
- other: Yes; concurrent solvent, positive and negative controls were run
- Details on study design:
- SOLUBILITY/PRECIPITATION ASSAY
The limit of test material solubility was determined by visual observation. Compound solubility was determined in solvent. In addition, the solutions were watched closely when added to the experiment tube (as the test material may precipitate upon addition to the assay tube mixtures).
ASSAYS
WORKING ASSAY BUFFER PREPARATION
Summary of Assay Conditions
- Source of receptor: Rat uterine cytosol
- Concentration of radioligand: 1 nM
- Concentration of receptor: Sufficient to bind 10 to 15 % of radioligand
- Temperature: 4 ± 2 °C
- Composition of assay buffer: Tris 10 mM (pH 7.4), EDTA 1.5 mM, Glycerol 10 % (v/v), Protease Inhibitor 0.5 % (v/v) and DTT 1 mM.
On the day of assay, the Working Assay Buffer, or TEDG+PI buffer (10 mM Tris, 1.5 mM EDTA, 1 mM DTT, 0.5 % Protease Inhibitor (v/v), 10 % glycerol, pH 7.4) was prepared using the 2X TEG buffer.
[³H]-17Β-ESTRADIOL PREPARATION
[³H]-17β-estradiol was prepared on the day of assay. The specific activity was adjusted for decay over time prior to performing dilutions. The specific activity was calculated on the day of the assay using the following equation:
SAadjusted (Fraction Isotope Remaining) = SA * e^(-Kdecay*Time)
Where:
SA = the specific activity on the packaging date
Kdecay = the decay constant for tritium (equal to 1.54 x 10^-4/day).
Time = days since the date on the stock bottle from the manufacturer.
The [³H]-17β-estradiol was diluted with TEDG + PI buffer so that each assay tube contained 1 nM final concentration of [³H]-17β-estradiol using the following procedure:
The specific activity was converted from Ci/mmole to nM. If SA = X Ci/mmole, and Y = concentration of radiolabel, then X Ci/mmole was converted to nM and the SA activity adjusted for decay over time by the following conversion:
(Y mCi/mL / X Ci/mmole) * 1 Ci/1000 mCi * 10^6 nmole/mmole * 1000 mL/L = (Y/X) * 10^6 nM
A 50 nM diluted stock of the [³H]-17β-estradiol was prepared so that 10 μL in a total volume of 500 μL per assay tube will give a final concentration of 1 nM. The 50 nM [³H]-17β-estradiol was kept on ice.
ASSAY PREPARATIONS
12 x 75 mm siliconised tubes were used for the assay. A master mixture of radioligand and buffer was prepared. An example is 153 tubes are required for a run that includes the solvent control, three standards, and three unknowns. Trace tubes are also required.
INDIVIDUAL TUBES
For the assay tubes, 390 μL of the master mixture was added and kept on ice. For the trace tubes, 50 μL (1 nM [³H]-17β-estradiol) final was added directly to 10 mL of scintillation fluid in scintillation vials and counted immediately. The standards, weak positive, negative and test chemicals were prepared and added to the assay tubes. 10 μL of chemical was added per tube. After all chemicals were added to the tubes, 100 μL of cytosol was added to each tube for a final volume of 500 μL. The temperature of the tubes and contents were kept at 4 ± 2 °C prior to the addition of the cytosol. The assay tubes were vortexed after additions and incubated at 4 ± 2 °C for 16 to 20 hours on a rotator.
Competitive Binding Assay Additions: 10 µL unlabelled 17β-estradiol, weak positive control, negative control, or test material; 390 µL master mixture (TEDG + PI assay buffer + [³H]-17β-estradiol; and 100 µL uterine cytosol (diluted to appropriate protein concentration). The total volume in each assay tube was 500 µL.
SEPARATION OF BOUND [³H]-17Β-ESTRADIOL FROM FREE [³H]-17Β-ESTRADIOL
The ER assay tubes were removed from the rotator and placed in an ice-water bath. A repeating pipette was used to add 250 μL of ice cold HAP slurry (60 % in TEDG + PI) to each assay tube. The tubes were vortexed for approximately 10 seconds at 5 minute intervals for a total of 15 minutes with tubes remaining in the ice-water bath between vortexing. Following the vortexing step, 2 mL of the cold (4 ± 2 °C) TEDG + PI buffer was added, quickly vortexed, and centrifuged at 4 ± 2 °C for approximately 10 minutes at 1000 x g. After centrifugation, the supernatant containing the free [³H]-17β-estradiol was immediately decanted and discarded.
The HAP pellet contained the estrogen receptor bound [³H]-17β-estradiol. Approximately 2 mL of ice-cold TEDG + PI buffer was added to each tube and vortexed to re-suspend the pellet. The tubes were centrifuged again at 4 ± 2 °C for approximately 10 minutes at 1000 x g.
The supernatant was quickly decanted and discarded. The wash and centrifugation steps were repeated once more. After the final wash, the supernatant was decanted. The assay tubes were allowed to drain briefly for approximately 30 seconds.
EXTRACTION AND QUANTIFICATION OF [³H]-17Β-ESTRADIOL BOUND TO ER
Approximately 1.5 mL of absolute ethanol was added to each assay tube. The tubes were allowed to sit at room temperature for approximately 15 to 20 minutes, vortexing for approximately 10 seconds at 5-minute intervals. The assay tubes were centrifuged for approximately 10 minutes at 1000 x g. A 1 mL aliquot was pipetted, taking care to avoid the centrifuged pellet, into a 20 mL scintillation vial containing approximately 10 mL scintillation cocktail (Perkin Elmer Opti-Fluor). The vial was capped and shaken. The vials were placed in a scintillation counter (Perkin Elmer Tri-Carb 2910TR Liquid Scintillation Analyzer Model B2910) and each vial was counted for at least one minute with quench correction for determination of DPMs per vial.
Standards (³H, 14C and background) were used to verify accurate counting, and the liquid scintillation analyzer has an enhanced Instrument Performance Assessment (IPA) for monitoring efficiencies, backgrounds, E2/B and Chi-square values for ³H and 14C over the life of the instrument. - Positive control:
- POSITIVE AND NEGATIVE CONTROL PREPARATION
Octyltriethoxysilane was the negative control chemical. A 100mM stock was prepared in DMSO and serially diluted. The final concentration range for the negative control was 1 x 10^-10 to 1 x 10^-3 M with DMSO kept at approximately 2 %.
The weak positive control was 19-norethindrone. A 10mM stock was prepared in DMSO and serially diluted. The final concentration range tested for the weak positive control was from 1 x 10^-8.5 to 1 x 10^-4 M with DMSO kept at approximately 2 %.
The reference standard (17β-estradiol) was included to ensure that the run was properly performed and to allow an assessment of variability in the conduct of the assay across time.Final concentrations of unlabelled 17β-estradiol ranged from 1 x 10^-7 to 1 x 10^-11 M. Fresh 50 μM 17β-estradiol stock was prepared and serial dilutions of the reference standard were in DMSO. - Details on results:
- CONCENTRATION RANGE FOR THE TEST MATERIAL
The final concentrations of the test material to assess precipitation were 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10-3 M.
The suitable top concentration for use in the binding assays was 10^-4 M; there was precipitation observed at 10-3 M. The final concentrations tested in the binding assays were: 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 M for the first two valid independent runs and 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 M for the third valid independent run.
BINDING ASSAY RESULTS
In three independent runs of the assay, the mean specific binding was > 75 % at every soluble concentration of the test material tested, with a maximal displacement of the radioligand from the ERs of 14.8 % (first independent run).
In all three independent runs, the mean specific binding was > 80 % for the negative control octyltriethoxysilane. In the first independent run, the mean specific binding was > 75 % at every soluble concentration tested for the test material. The mean specific binding for the test material at 10^-3 M was 54.8 % of control, however there was precipitation observed at this concentration so the data was not evaluated. The weak positive control 19-norethindrone had a LogIC50 of -5.6 M while the LogIC50 of 17β-estradiol was -9.0 M.
In the second independent run, the mean specific binding was > 75 % at every soluble concentration tested for the test material. The mean specific binding for the test material at 10^-3 M was 66.3 % of control, however there was precipitation observed at this concentration so the data was not evaluated. The weak positive control 19-norethindrone had a LogIC50 of -5.5 M while the LogIC50 of 17β-estradiol was -9.6 M.
Finally, in the third independent run, the test material was tested at 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 M and the mean specific binding was > 75 % at every concentration tested. The weak positive control 19-norethindrone had a LogIC50 of -5.6 M while the LogIC50 of 17β-estradiol was -9.1 M.
The mean relative binding affinity, or RBA (calculated by dividing the LogIC50 of the control/test material by the LogIC50 of the positive control 17β-estradiol) was 0.6 for 19-norethindrone. There was no RBA to be calculated for the test material.
The test material was classified as “non-interacting” in all three independent runs and thus has a final classification of “non-interacting.”
BINDING ASSAY ACCEPTANCE CRITERIA
In all three independent runs of the assay, increasing concentrations of unlabelled 17β-estradiol displaced [³H]-17β-estradiol from the receptor in a manner consistent with one-site competitive binding, and the ligand depletion was held below 15 %. Also, the solvent did not alter the assay sensitivity or reliability. The negative control, octyltriethoxysilane, did not displace more than 25 % of the radioligand from the ERs (maximum displacement of 19.6 %).
Finally, the data were within the acceptable ranges specified with the following exceptions:
In the first run of the assay, the bottom plateau level for 19-norethindrone was slightly more than the specified range (bottom plateau level = 2 %; compared to the specified range of -5 to 1 %).
In the first run of the assay, the hill slope for 19-norethindrone was slightly less than the specified range (bottom plateau level = -1.3 %; compared to the specified range of -0.7 to -1.1 %).
These deviations were minor and not considered to reflect true deviation from the suggested ranges outlined in the guideline. Therefore, both independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be definitive.
10^-3 M test material data for the first two valid independent runs were excluded from evaluation and interpretation due to precipitation. - Conclusions:
- The test material was classified as “non-interacting” in all three independent runs and thus has a final classification of “non-interacting.”
- Executive summary:
A study was conducted to evaluate the ability of the test material to interact with the estrogen receptors (ERs) isolated from the rat uterus in accordance with the standardised guideline US EPA OPPTS 890.1250 under GLP conditions.
Preliminary assessments of precipitation were conducted in order to identify a suitable top concentration for use in the binding assays. The suitable top concentration for use in the binding assays was 10^-4 M. There was mild precipitation observed at 10^-3 M.
The final concentrations tested in the binding assays were: 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 M for the first two valid independent runs and 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5 and 10^-4 M for the third valid independent run.
Three independent runs of the binding assay were conducted. All concentrations were tested in replicates of 3. In addition, 3 replicates (solvent (DMSO) control tubes) were prepared to assess total binding. These replicates included the radioligand, cytosol (containing the ERs) and solvent but without the competitor. The total binding tubes allowed for the identification of maximal binding of [³H]-17β-estradiol. Non-specific binding (NSB) was also assessed in replicates of 3 by determining the [³H]-17β-estradiol bound in the presence of 100-fold excess unlabelled 17β-estradiol. Data was background and NSB subtracted, normalised to total binding and presented as % Specific Binding. Finally, total radioligand added was determined (n = 6), which was used to calculate the percentage of total radioligand added to the tube that is bound to ERs. The duration of incubation was 16 to 20 hours. A complete concentration response curve for the negative control (NC) octyltriethoxysilane, and weak positive control 19-norethindrone, was run each time the binding assay was performed.
In all three independent runs, the mean specific binding was > 80 % for the negative control octyltriethoxysilane. In the first independent run, the mean specific binding was > 75 % at every soluble concentration tested for the test material. The mean specific binding for the test material at 10^-3 M was 54.8 % of control, however there was precipitation observed at this concentration so the data was not evaluated. The weak positive control 19-norethindrone had a LogIC50 of -5.6 M while the LogIC50 of 17β-estradiol was -9.0 M.
In the second independent run, the mean specific binding was > 75 % at every soluble concentration tested for the test material. The mean specific binding for the test material at 10^-3 M was 66.3 % of control, however there was precipitation observed at this concentration so the data was not evaluated. The weak positive control 19-norethindrone had a LogIC50 of -5.5 M while the LogIC50 of 17β-estradiol was -9.6 M.
Finally, in the third independent run, the mean specific binding was > 75 % at every concentration tested. The weak positive control 19-norethindrone had a LogIC50 of -5.6 M while the LogIC50 of 17β-estradiol was -9.1 M.
The mean relative binding affinity, or RBA (calculated by dividing the LogIC50 of the control/test material by the LogIC50 of the positive control 17β-estradiol) was 0.6 for 19-norethindrone. There was no RBA to be calculated for the test material.
Under the conditions of this study, the test material was classified as “non-interacting” in all three independent runs and thus has a final classification of “non-interacting.”
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 April 2011 to 25 October 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1300
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 455
- Deviations:
- no
- GLP compliance:
- yes
- Type of method:
- in vitro
- Endpoint addressed:
- other: Estrogen receptor transcriptional activation
- Species:
- human
- Strain:
- other: Cell line HeLa-9903
- Details on test animals or test system and environmental conditions:
- CELL LINE
The stably transfected hERα-HeLa-9903 cell line was used in this study. The cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, 7-6-8 Asagi Saito, Ibaraki-shi, Osaka 567-0085, Japan.
The cell line was certified to be free of mycoplasma. The cells used in this study were passage 32 (rangefinder) to passages 33 and 34 (two valid independent runs prior to seeding into plates.
CELL CULTURE AND PLATING CONDITIONS
Cells were maintained in Eagle’s Minimum Essential Medium (EMEM) without phenol red, supplemented with 60 mg/L of Kanamycin (antibiotic) and 10 % dextran-coated-charcoal-treated foetal bovine serum (DCC-FBS), in a CO2 incubator (5 % CO2) at 37 ± 1 °C. When the cells reached 75 to 90 % confluency, they were sub-cultured at 10 mL of 0.4 x 10^5 to 1 x 10^5 cells/mL. The cells were suspended with 10 % DCC-FBS in EMEM and plated into wells of a 96 well cell culture plate at a density of ~1 x 10^4 cells/100 µL/well. The cells were then placed into a 5 % CO2 incubator at 37 ± 1 °C for at least 3 hours prior to chemical exposure. - Route of administration:
- other: in vitro
- Vehicle:
- DMSO
- Details on exposure:
- PREPARATION OF STOCK SOLUTIONS
The reference chemicals and test material were dissolved in DMSO then serially diluted as appropriate in DMSO before further dilution in medium to prepare 2x concentrated stock solutions. When added to the cell culture plates, the stock solutions would yield the final serial concentrations as required for the reference chemicals and as determined for in the preliminary range finding assays for the test material.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material solutions with a concentration of up to 10^-4 M (the limit concentration for the assay) can be prepared.
- Concentration in vehicle: The final concentration of DMSO in the medium was held constant at 0.1 % (v/v). - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 24 ± 2 hours
- Frequency of treatment:
- Once
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0 other: M
- Dose / conc.:
- 0.001 other: M
- Dose / conc.:
- 0.01 other: M
- Remarks:
- Additional concentrations: 10^-8.5, 10^-7.5, 10^-6.5 and 10^-5.5 M
- No. of animals per sex per dose:
- All concentrations were tested in replicates of 6/plate. In addition, for each concentration, 2 replicates/plate were prepared that incorporated the hERα antagonist ICI 182,780. The test was conducted as a rangefinder and two valid independent runs.
- Control animals:
- other: Yes; concurrent solvent and positive controls were run
- Details on study design:
- CHEMICAL EXPOSURE AND ASSAY PLATE ORGANISATION
Following addition of the test material and reference substances to the cell culture plates, and after the three hour (minimum) post-seeding incubation, the plates were removed from the incubator and the media was aspirated. 75 μL of fresh media, followed by 75 μL of the 2x concentrated stock solutions were added to wells containing ~1 X 10^4 cells/well for a final volume of 150 µL/well.
After adding the reference chemicals/test material, the plates were incubated in a 5 % CO2 incubator at 37 ± 2 °C for 24 ± 2 h. All concentrations were tested in replicates of 6/plate. In addition, for each concentration, 2 replicates/plate were prepared that incorporated the hERα antagonist ICI 182,780. Replicates incorporating a hERα antagonist allow for the identification of non-specific (i.e., non-hERα-mediated) induction of the luciferase gene as true hERα-mediated induction is inhibited by addition of an antagonist whereas non-specific induction is not.
ASSAYS
CYTOTOXICITY ASSAY
Cell viability was monitored by a two-read propidium iodide (PI) uptake assay. PI is a light sensitive dye and all procedures were conducted under low light conditions. PI cannot cross the plasma membrane of intact and viable cells. Cells that are dead or dying have weakened plasma membranes which allow PI to enter the cytosol of the damaged cells. Once inside the cell, PI intercalates into DNA/RNA and yields a fluorescent signal. The intensity of the fluorescent signal is inversely proportional to cell viability, where a decrease in cell viability is detected by an increase in fluorescent signal. In the two-read procedure, the first read is taken immediately after full exposure to controls and test material is completed. This measures “background” fluorescence and indicates cell spontaneous death and control/test material-induced cytotoxicity. The cells are then lysed and a second read is taken, which indicates 100 % cell death. The first read is then subtracted from the second read. The results of the subtracted reads are directly proportional to the viability of the cells. The control and test material data are normalised to vehicle control to generate percent cell viability.
Cells were seeded as described above in a black-walled 96-well cell culture plate. The cells were exposed to the test chemicals in replicates of 6 (rows A-F) while the last 2 rows (G and H) received 125 μM digitonin as a positive control for cell death. Following chemical exposure, the growth medium was removed and 50 µL of a PI working solution (44 μM in phosphate buffered saline or cell culture medium was added to each well. Background fluorescence was evaluated by measuring fluorescence immediately on a Packard Fusion fluorescence plate reader at an excitation wavelength of 544 nm and an emission wavelength of 612 nm. Following this determination, 50 µL of a 2 % (v/v) Triton X-100 solution was added to each well and the plate was incubated at room temperature for a minimum of 15 minutes to fully lyse all cells in the wells before measuring fluorescence at the same wavelengths.
The background-corrected fluorescence was calculated for each well by subtracting the results of the first read from the results of the second read. The change in cell viability was determined by comparing treated wells to the vehicle control wells. A ≥20 % reduction in cell viability was considered evidence of cytotoxicity.
PRECIPITATION ASSAY
The limit of solubility was determined by visual inspection of the test material and controls after preparation of the final 1x dosing solutions in culture media. A sample of each 1x dosing solution was placed into a well of a clear 96-well plate and an endoscope was used to assess precipitation in each sample.
TRANSCRIPTIONAL ACTIVATION ASSAY
A luciferase assay was performed using the following reagents: Trisma Base, Magnesium Chloride, EDTA, Dithiothreitol, ATP, Coenzyme A, AMP, Luciferin, Glycerol , Triton-X100, Bovine Serum Albumin and CDTA.
PRELIMINARY RANGE FINDING
In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted.
These preliminary assays assessed precipitation and cytotoxicity at the following concentrations (up to the limit concentration of 10^-3 M): 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 M.
TRANSCRIPTIONAL ACTIVATION ASSAY DATA ANALYSIS AND INTERPRETATION
In order to determine the relative transcriptional activity as compared to the positive control (PC), 1 nM 17β-estradiol, the luminescence data from each plate were analysed according to the steps outlined below. Wells incorporating ICI 182,780 were analysed in an identical fashion to wells not incorporating ICI 182,780, except that the data were normalised by subtracting the mean value for the ICI 182,780-containing vehicle control (VC) wells.
1. Any cytotoxic concentrations were excluded from data analysis.
2. The mean value for the VC wells was calculated.
3. The mean value for the VC wells was subtracted from each well to normalise the data.
4. The mean value for the normalised PC wells was calculated.
5. The normalised value for each well was divided by the mean value of the normalised PC wells (with the normalised mean of the PC wells being defined as 100 % relative transcriptional activity). The final value for each well is the relative transcriptional activity for that well compared to the mean normalised PC response.
The data were then interpreted according to the following steps:
1. Where appropriate, LogPC50, LogPC10, LogEC50 and Hill slope values were calculated.
2. For the test material, the maximum response relative to the positive control (RPCMax) was determined. In each individual run of the transcriptional activation assay, if RPCmax was less than 10 %, the test material was considered to have given a negative response for hERα agonism.
3. For each individual run of the transcriptional activation assay, the acceptability of the data was evaluated using the following criteria:
- The mean normalised luciferase signal of the PC (1 nM 17β-estradiol) should be at least 4-fold that of the mean VC on each plate.
- The results of the 4 reference chemicals should be within the acceptable ranges.
4. If the acceptability criteria outlined above were met, that run of the transcriptional activation assay was considered to be valid.
5. The test material was considered negative if RPCMax <10 % in at least 2 valid runs of the transcriptional activation assay. The test material was considered positive if RPCMax ≥10 % in at least 2 valid runs of the transcriptional activation assay. If the results are not reproducible, a deciding third run would be performed. - Positive control:
- STABILITY OF THE CELL LINE
The stability of the cell line was monitored by the use of the following reference chemicals: 17β-estradiol, 17α-estradiol, 17α-methyltestosterone and corticosterone. A complete concentration response curve for each reference compound was run each time the transcriptional activation assay was performed and the LogPC50, LogPC10, LogEC50 and Hill slope values calculated and compared to the acceptable range values. - Details on results:
- CONCENTRATION RANGE FOR THE TEST MATERIAL
In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary assessments of precipitation and cytotoxicity were conducted.
The suitable top concentration of the test material for use in the transcriptional activation assays was 10^-6 M, based on excessive cytotoxicity (≥20 % reduction in cell viability) at concentrations ≥10-5 M identified in the range finder and both independent runs. The test material was also deemed to be insoluble at ≤10^-3 M. Precipitation was observed at this concentration.
The final concentrations of the test material tested in the transcriptional activation assays were: 10^-12, 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6 and 10^-5 M for the initial run and 10^-8.5, 10^-8, 10^-7.5, 10^-7, 10^-6.5, 10^-6, 10^-5.5 and 10^-5 M for the second run.
TRANSCRIPTIONAL ACTIVATION ASSAY RESULTS
Three independent runs of the transcriptional activation assay were conducted because in the first run, significant cytotoxicity issues were observed across all controls and the test material. Historical data shows no toxicity with controls, so the first run was determined to be a bad run.
In two valid independent runs of the assay, the test material did not result in an increase in luciferase activity at any of the concentrations tested as the RPCmax values in both independent runs of the assay were <10 % (Mean highest induction values of 3.6 ± 3.1 % and -1.7 ± 3.8 % in the first and second valid independent runs of the assay, respectively).
The test material is not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.
TRANSCRIPTIONAL ACTIVATION ASSAY ACCEPTANCE CRITERIA
In both valid independent runs of the assay, the mean luciferase activity of the PC (1 nM 17β-estradiol) was greater than 4-fold that of the mean luciferase activity of the VC on each plate. In addition, in both independent runs of the assay the LogPC50, LogPC10, LogEC50 and Hill slope values for the 4 reference compounds (17β-estradiol, 17α- estradiol, 17α-methyltestosterone and corticosterone) were within the acceptable ranges, with the following minor exceptions:
- In the first valid run of the assay, the LogPC10 for 17α-estradiol was marginally lower than the specified range (LogPC10 = -11.0; compared to the specified range of -10.7 to -9.3).
- In the first valid run of the assay, the LogPC50 for 17α-estradiol was marginally lower than the specified range (LogPC50 = -9.9; compared to the specified range of -9.6 to -8.1).
- In the first valid run of the assay, the LogEC50 for 17α-estradiol was marginally lower than the specified range (LogEC50 = -10.0; compared to the specified range of -9.6 to -8.4).
- In the first valid run of the assay, the Hill Slope for 17β-estradiol was marginally greater than the specified range (Hill Slope = 1.6; compared to the specified range of 0.7 to 1.5).
- In both valid runs of the assay, the LogPC50 values for 17β-estradiol were marginally lower than the specified range (LogPC50 = -11.5 and -11.7 for runs 1 and 2, respectively; compared to the specified range of -11.4 to -10.1).
- In both valid runs of the assay, the LogEC50 values for 17β-estradiol were marginally lower than the specified range (LogEC50 = -11.4 and -11.5 for runs 1 and 2, respectively; compared to the specified range of -11.3 to -10.1).
- In both valid runs of the assay, the LogPC10 values for 17α-methyltestosterone were lower than the specified range (LogPC10 = -10.7 and -9.7 for runs 1 and 2, respectively; compared to the specified range of -8.0 to -6.2).
- In both valid runs of the assay, the LogPC50 values for 17α-methyltestosterone were lower than the specified range (LogPC50 = -9.6 and -8.8 for runs 1 and 2, respectively; compared to the specified range of -6.0 to -5.1).
These deviations were minor and not considered to reflect true deviation from the suggested ranges outlined in the OPPTS guideline. Therefore, both independent runs of the assay were considered to have met the assay acceptance criteria and were considered to be valid. - Conclusions:
- The test material is not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.
- Executive summary:
A study was conducted to evaluate the ability of the test material to act as an agonist of human estrogen receptor alpha (hERα) using the hERα-HeLa-9903 cell line in accordance with the standardised guidelines US EPA OPPTS 890.1300 and OECD 455 under GLP conditions.
Preliminary assessments of cytotoxicity and precipitation were conducted in order to identify a suitable top concentration of the test material for use in the transcriptional activation assays.
The final concentrations of the test material tested in the transcriptional activation assays were: 10^-12, 10^-11, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6 and 10^-5 M for the first valid run and 10^-8.5, 10^-8, 10^-7.5, 10^-7, 10^-6.5, 10^-6, 10^-5.5, and 10^-5 M for the second valid run.
All concentrations were tested in replicates of 6/plate. In addition, for each concentration, 2 replicates/plate were prepared that incorporated the hERα antagonist ICI 182,780. Replicates incorporating the hERα antagonist allow for the identification of non-specific (i.e., non-hERα-mediated) induction of the luciferase gene. The duration of exposure was 24 h. A complete concentration response curve for each of 4 reference compounds (17β-estradiol, 17α-estradiol, corticosterone and 17α-methyltestosterone) was run each time the transcriptional activation assay was performed.
The suitable top concentration of the test material for use in the transcriptional activation assays was 10^-6 M, based on excessive cytotoxicity (≥20 % reduction in cell viability) at concentrations≥10^-5 M. The test material was also deemed to be insoluble at≤10^-3 M.
In two independent runs of the transcriptional activation assay, the test material did not result in an increase in luciferase activity at any of the viable concentrations tested (RPCmax<10 %).
Under the conditions of this study, the test material is not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 April 2011 to 12 May 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OPPTS 890.1350
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 229
- Deviations:
- no
- Principles of method if other than guideline:
- Although this study was conducted in a fish species, it was considered relevant to include it in this section as part of the overall discussion of endocrine effects.
- GLP compliance:
- yes
- Type of method:
- in vivo
- Endpoint addressed:
- other: To assess the potential endocrine activity of the test material in fathead minnow (Pimephales promelas) exposed under flow-through conditions
- Species:
- other: fish
- Strain:
- other: Pimephales promelas
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ORGANISM
The fathead minnow used during this study (Lot No. 10A129) were obtained from a laboratory supply of reproductively mature animals (approximately 24 weeks old), in spawning condition. Prior to testing, spawning groups consisting of two males and four females were held in a 39 x 20 x 25 cm (L x W x H) glass aquarium with an overflow drain 13 cm from the bottom under a photoperiod of 16 hours of light and 8 hours of darkness. Substrates were provided for the females to deposit the adhesive eggs. The substrate was constructed from 4-inch diameter appropriately aged PVC pipe. Seven cm sections of pipe were cut and sectioned in half lengthwise to produce “arches” under which spawning took place. Each vessel contained three spawning substrates. Water flowing to the culture unit was from the same source as the dilution water used during the exposure.
The water which flowed into this holding tank was characterised as having total hardness and total alkalinity ranges as calcium carbonate (CaCO3) of 38 to 74 mg/L and 20 to 27 mg/L, respectively, a pH range of 6.8 to 7.4 and a specific conductance range of 240 to 390 μmhos/cm. Fish used during the definitive exposure were maintained under these conditions for 23 days prior to testing. The temperature in the holding tanks ranged from 24 to 26 °C during this period.
Fish did not receive treatment for disease in the two-weeks prior to the spawning qualification period preceding the test, or during the exposure period. There were 10 mortalities observed (5 %) in the test fish population during the 23-days period prior to testing.
Prior to test initiation and throughout the exposure period, the fish were fed a measured amount twice daily with frozen brine shrimp.
SELECTION AND WEIGHING OF TEST FISH
Prior to adding fish to the pre-exposure system, subsamples of both male and female fish in the test population were weighed. A representative sample of the male (N = 20) and female (N = 20) fish from the test population selected for the test material exposure had a mean wet weight of 3.7 g (range 3.0 to 4.6 g or ± 20 % of the mean) and 1.7 g (range 1.4 to 2.0 g or ± 20 % of the mean), respectively. - Route of administration:
- other: Fish were exposed in dilution water under flow-through conditions
- Vehicle:
- acetone
- Details on exposure:
- DILUTION WATER
The dilution water used during this study was on-site well water which was pumped into an epoxy-coated concrete reservoir and aerated prior to use. During the study, weekly characterisation of the well water established total hardness and alkalinity ranges as CaCO3 of 38 to 78 mg/L and 26 to 29 mg/L, respectively, a pH range of 6.9 to 7.9, and a specific conductance range of 350 to 390 μmhos/cm.
STOCK PREPARATION
A 1.5 mg a.i./mL diluter stock solution was prepared prior to test initiation and weekly thereafter by placing, for example, 0.0867 g of the test material (0.0772 g as active ingredient) in a 50.0 mL volumetric flask and bringing it to volume with acetone. The resulting stock solution was observed to be clear and colourless with no visible undissolved test material following preparation.
A 0.43 mL/mL solvent stock solution was prepared by diluting 433 mL of acetone to a final volume of 1.0 L with purified reagent water. The resultant solvent stock solutions were observed to be clear and colourless following preparation. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to the start of the definitive exposure, samples from one replicate of each treatment level and the controls were collected and analysed. Samples of the stock solution were also analysed during the pre-test period. The diluter stock solution was analysed at each sampling interval during definitive testing. Results of the pre-test analyses were used to judge whether the diluter was functioning properly.
During the fish screening study, water samples were removed from all replicates (respectively, A, B, C and D) of each treatment level and the controls at day 0 (test initiation) and day 5, in order to confirm proper diluter function. On test days 14 and 21 (test termination), samples were removed from two alternating replicates (A, C and B, D, respectively). Water samples were removed from a point approximately midway between the surface, bottom and sides of each test vessel and analysed immediately.
Since the sampling intervals were not equally spaced (e.g., 0, 5, 14 and 21 days) and exposure concentrations decreased slightly over the course of the exposure, a time-weighted average was calculated for each treatment level.
Three quality control (QC) samples were prepared and analysed with the each set of study samples. These QC samples were prepared in dilution water at concentrations similar to the treatment levels tested. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
All exposure solutions and QC samples were analysed for the test material by using liquid chromatography/mass spectrometry (LC/MS/MS) based on methodology validated at the testing facility. The method validation study was conducted prior to the initiation of the definitive exposure and established an average recovery of 109 ± 5.03 % for the test material from aged FETAX solution. Defined limits for acceptance of quality control sample performance in subsequent studies were set at 80 to 120 %. Conditions and procedures used throughout the analysis of exposure solutions and QC samples during this study were similar to those used in the method validation study.
ANALYTICAL METHOD
A method validation was performed to quantify the amount of test material present in aged FETAX solution. Recovery samples were initially diluted with acetonitrile to a final composition of 50:50 acetonitrile:aged FETAX solution (v:v). The mid and high concentration recovery samples were further diluted into the calibration standard range with 50:50 acetonitrile:aged FETAX solution (v:v). All samples were analysed by liquid chromatography/mass spectrometry (LC/MS/MS).
This method validation was conducted by fortification of aged FETAX solution with test material at concentrations of 0.0500, 2.00 and 50.0 μg a.i./L. Recoveries averaged 109 ± 5.03 %, with a limit of quantitation of 0.0182 μg a.i./L. Defined limits for acceptance of quality control sample performance in subsequent studies were set at 80 to 120 %.
Concentrations were adjusted for the purity of the test material and are presented as active ingredient (a.i.).
- Preparation of Stock Solutions
A 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0562 g of the test material (0.0501 g as active ingredient) in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. Two secondary stock solutions (1.00 and 10.0 mg a.i./L) were prepared by placing 0.0500 and 0.500 mL, respectively, of the 1.00 mg a.i./mL primary stock solution in individual 50.0-mL volumetric flasks and bringing each to volume with acetonitrile. The 1.00 and 10.0 mg a.i./L secondary stock solutions were used to fortify the mid- and high-level recovery samples, respectively. A 10.0 μg a.i./L sub-stock solution was prepared by placing 0.100 mL of the 1.00 mg a.i./L secondary stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution. This 10.0 μg a.i./L substock solution was used to fortify the low-level recovery samples.
An additional 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0562 g of the test material (0.0501 g as active ingredient) in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. A 10.0 mg a.i./L secondary stock solution was prepared by placing 0.500 mL of the 1.00 mg a.i./L primary stock solution in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. A 100 μg a.i./L sub-stock solution was prepared by placing 0.100 mL of the 10.0 mg a.i./L secondary stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution.
Another sub-stock solution (10.0 μg a.i./L) was prepared by placing 1.00 mL of the 100 μg a.i./L sub-stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution. The 10.0 and 100 μg a.i./L sub-stock solutions were used to prepare calibration standards.
All primary and secondary stock solutions were stored in a refrigerator in glass amber bottles fitted with Teflon®-lined caps. All sub-stock solutions were prepared fresh daily and discarded after use.
- Preparation of Calibration Standards
Calibration standards were prepared in 50:50 acetonitrile:aged FETAX solution using the 10.0 and 100 μg a.i./L sub-stock solutions to yield concentrations of 0.0100, 0.0200, 0.0500, 0.200, 0.500 and 1.00 μg a.i./L.
- Sample Fortification and Preparation
The recovery samples were prepared by fortifying aged FETAX solution with test material to obtain concentrations of 0.0500, 2.00 and 50.0 μg a.i./L. The low-level recovery samples were fortified using the 10.0 μg a.i./L sub stock solution. The mid- and high-level recovery samples were fortified using the 1.00 and 10.0 mg a.i./L secondary stock solutions, respectively. An aliquot (5.00 mL) of each recovery sample were diluted to a final volume of 10 mL with acetonitrile. The mid- and high-level recovery samples (1.00 and 0.0500 mL, respectively) were further diluted into the calibration standard range (to a final volume of 10 mL) with 50:50 acetonitrile:aged FETAX solution (v:v). Three recovery samples were prepared for each concentration. In addition, three recovery samples were left unfortified to serve as controls and were diluted in a similar fashion as the low-level recovery samples.
- Instrumental Conditions
The LC/MS/MS analysis was conducted utilising the following instrumental conditions:
LC parameters:
Column: Phenomenex Synergi MAX-RP, 4 μm, 2.0 mm x 50 mm
Mobile Phase A: 3 mM ammonium acetate in 10:90 acetonitrile:purified reagent water
Mobile Phase B: 3 mM ammonium acetate in 90:10 acetonitrile:purified reagent water
Gradient (flow rate 300 µL/min): At 0.0 and 1.0 minutes 65 % A, 35 % B; at 2.50 and 5.50 minutes 2 % A, 98 % B; and at 5.70 and 9.0 minutes 65 % A, 35 % B
Injection volume: 100 μL
Column oven: 40 °C
Retention Time: approximately 4.3 minutes
MS parameters:
Instrument: MDS Sciex 4000Q TRAP® mass spectrometer equipped with an MDS Sciex 4000Q TRAP® ESI Turbo V Source
Ionisation mode: positive
Q1/Q3 masses: 368.00/231.00 amu
Dwell time: 600 milliseconds
Source temperature: 450 °C
Scan type: MRM
- Preparation of Standard Curve
Two sets of calibration standards were analysed with each recovery sample set; one set prior to analysis of the recovery samples, and the second set immediately following the analysis of the recovery samples. Injection of samples and calibration standards onto the LC/MS/MS system was performed by programmed automated injection.
- Calculations
A calibration curve was constructed by plotting the analyte concentration (μg a.i./L) in the calibration standards against the peak area of the calibration standards. The equation of the line (equation 1) was algebraically manipulated to give equation 2. The concentration of the test material within each recovery sample was determined using the regression coefficients from the quadratic equation, the peak area of the recovery sample, and the dilution factor. Equations 2 and 3 were then used to calculate measured concentrations and analytical results.
Equation 1: y = ax² + bx +c
Equation2: DC (x) = (-b + √(b² - 4aC)) / 2a
Equation 3: A= DC x DF
where:
y = detector response (peak area) for analyte
a, b and c = regression constants
DC (x) = detected concentration (μg a.i./L) in the sample
C = constant c minus the peak area; C = (c - y)
DF = dilution factor (the final sample volume divided by the original sample volume)
A = concentration of the analyte in the original sample
The limit of quantitation (LOQ) was calculated using the following:
Area(MIN) = A(LS)
LOQ(inst) = -b + √(b² - 4aC)
LOQ = LOQ(inst) x DF(ctrl)
where:
AreaMIN = mean detector response (peak area) of the low concentration calibration standard (two injections)
ALS = mean detector response (peak area) of the low concentration calibration standard (two injections)
a, b, c = regression constants
C = regression constant c – Area(MIN); C = (c - Area(MIN))
LOQ(inst) = limit of quantitation on the instrument
DF(ctrl) = dilution factor of the control samples (smallest dilution factor used)
LOQ = limit of quantitation reported for the analysis - Duration of treatment / exposure:
- 21 days
- Frequency of treatment:
- continuous in dilution water
- Dose / conc.:
- 0.2 other: µg a.i./L (nominal)
- Dose / conc.:
- 2 other: µg a.i./L (nominal)
- Dose / conc.:
- 20 other: µg a.i./L (nominal)
- No. of animals per sex per dose:
- Each replicate tank contained four female and two male fish
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- Details on study design:
- TEST CONDITIONS
The aquaria were positioned using a random block design in two (upper and lower) water baths containing circulating water designed to maintain the test solution temperatures at 25 ± 1 °C. Dissolved oxygen concentrations were maintained at > 60 % of saturation. Illumination was provided by Sylvania Oktron® fluorescent bulbs centrally located above the test aquaria.
Sixteen hours of light at 77 to 100 footcandles (830 to 1100 lux) at the exposure solution surface was provided each day. The diluter system was enclosed with black polyethylene curtains to prevent disturbance of spawning and to minimise the interference of laboratory lighting with the intended photoperiod.
EXPOSURE SYSTEM
The exposure system consisted of a 2000-mL intermittent-flow proportional diluter and a two-tiered water bath, consisting of an upper and a lower level water bath (one positioned over the other). The exposure system was designed to provide three concentrations of the test material, a dilution water control and a solvent (acetone) control to four replicate exposure aquaria.
Prior to test initiation, a Harvard Apparatus Pump in conjunction with a 50-mL Glenco® gas-tight syringe was calibrated to deliver 0.02886 mL/cycle of the 1.5 mg a.i./mL diluter stock solution to the diluter's mixing chamber, which also received 2.2 L of dilution water per cycle. The mixing chamber of the diluter was positioned over a magnetic stir plate. The continuous stirring aided the solubilisation of the test material with the dilution water. The solution contained in the mixing chamber constituted the highest nominal test concentration (20 μg a.i./L) and was subsequently diluted to provide the remaining nominal exposure concentrations (2.0 and 0.20 μg a.i./L). Flow-splitting chambers were used to promote further mixing and to equally distribute the test solutions through glass delivery tubes to the replicate exposure aquaria. The negative control consisted of dilution water only.
The concentration of acetone in the solution in the mixing chamber and the high test concentration constituted the highest acetone concentration (0.013 mL/L). An FMI Pump was calibrated to deliver 0.294 mL/cycle of the solvent stock solution (0.43 mL/mL) to 9.8 L of dilution water per cycle which was subsequently delivered to the solvent control and treatment vessels. The acetone concentration in the solvent control and the treatment levels was 0.013 mL/L, which was equal to that of the high test concentration.
Calibration of the diluter system was confirmed prior to test initiation by measuring delivery volumes of the chemically-dosed solutions and the dilution water. The function of the diluter (e.g., dilution water flow rate, solvent flow rate, stock solution consumption) was monitored daily and a visual check was performed twice daily. In addition, analysis of exposure solutions for test material concentration was also used to verify proper operation of the diluter system.
Analysis of exposure solutions for the test material was performed on test days 0, 5, 14 and 21. The exposure system was operating properly for two days prior to study initiation to allow equilibration of the test material in the diluter apparatus and exposure aquaria. Test aquaria were labelled to identify the nominal test material concentration and designated replicate.
The exposure system and exposure aquaria were constructed of glass, silicone sealant and nylon. Each 18-L test aquarium measured 39 x 20 x 13 cm with a 13-cm high side drain that maintained a constant exposure solution volume of approximately 10 L.
Flow-splitting chambers were used between the diluter cells and the four replicate test vessels to promote mixing of the test material solution, solvent solution and diluent water, and to equally split the test solution between the test vessels. During the test, 0.5 L per cycle of test solution was delivered to each aquarium at a rate of approximately 200 cycles per 24 hours. This flow provided turnover rate of 9.7 volume replacements every 24 hours, or a 90 % replacement time of 5 hours.
Chemical cleaning of the diluter system and aquaria was performed prior to test initiation. During the 21-day study, exposure aquaria were brushed and siphoned twice per week to remove excess food and faecal matter. At a minimum, the diluter mixing chamber, chemical cells and splitters were cleaned weekly, and delivery tubing was cleaned as necessary.
PRE-EXPOSURE PHASE
Four females and two males were impartially assigned to each of the 36 pre-exposure vessels 23 days prior to test initiation. The additional exposure chambers were set up during the pre-exposure phase to account for a lack of spawning in some chambers and/or mortality during this phase. The pre-exposure phase was conducted under test conditions identical to those used during the chemical exposure. The animals were fed frozen brine shrimp (Artemia) twice daily during this phase. Each group was monitored daily for active spawning and fecundity data was collected. During this phase, suitability for testing was established when regular spawning occurred in each replicate chamber at least twice in the immediate 7-day period preceding test initiation or when an egg production rate of > 15 eggs/female/day/replicate was achieved. The spawning groups selected for the exposure met these criteria after a 23-day pre-exposure phase.
CHEMICAL EXPOSURE
Once successful spawning was established during the pre-exposure phase, the 20 spawn groups with the greatest number of eggs/female/day were added to the exposure system using a random block distribution which initiated the chemical exposure. The exposure was maintained for a period of 21 days. Each replicate tank contained four female and two male fish. During the exposure period, the appearance of the fish, behaviour and fecundity were assessed daily. At test termination, fish were anaesthetised by transfer to a buffered solution of MS-222, measured for total length and wet weight. Blood samples were removed for vitellogenin (VTG) analysis. The gonads were also removed and weighed for gonadosomatic index (GSI) determination and histological analyses. Fish were preserved for subsequent tubercle scoring and histopathology.
WATER QUALITY MEASUREMENTS
At test initiation and weekly thereafter, total hardness and total alkalinity were measured and recorded in one replicate of one treatment level and the control alternating between treatment levels and replicate vessels (A, B, C and D) each week. Dissolved oxygen, pH and temperature were recorded in each concentration and control vessel at test initiation and subsequently in alternating replicates on a daily basis. Test solution temperature was continuously monitored during the exposure period in the upper (solvent control replicate A) and lower (solvent control replicate C) water baths.
STATISTICAL ANALYSES
All statistical analyses were conducted at the 95 % level of certainty except in the case of Shapiro-Wilks' and Bartlett's Tests, in which the 99 % level of certainty was applied. The 99 % level of certainty is preferred for qualifying tests. The following procedures were used:
1. Fisher’s Exact Test (U.S. EPA, 2002) was conducted to statistically compare control to the solvent control data. For this study, no significant difference was determined between the control and solvent control data for any of the endpoints. Therefore, the control and solvent control data were pooled for comparison to the treatment levels for all endpoints.
2. Shapiro-Wilks' Test for normality (U.S. EPA, 2002) was conducted and compared the observed sample distribution with a normal distribution. The assumption that observations are normally distributed must be validated before subsequent analyses, following parametric procedures, can be performed. If the data is not normally distributed, then a non-parametric procedure is used for subsequent analyses.
3. As a check on the assumption of homogeneity of variance, implicit in parametric statistics, data for each endpoint were analysed using Bartlett's Test (U.S. EPA, 2002).
4. For endpoints that met the assumptions for normality and homogeneity, the performance of organisms exposed to each treatment level was compared with the performance of the control using Dunnett's Multiple Comparison Test (U.S. EPA, 2002). A nonparametric procedure, Jonckheere-Terpstra Step-Down Test (U.S. EPA, 2002), was also used for comparison when data did not meet the assumptions for normality and/or homogeneity of variance.
CETIS™ Version 1.8.0 (Ives, 2009) was used to perform the statistical computations.
Histopathology results were evaluated qualitatively. - Examinations:
- TEST TERMINATION
- Gonad Size and Histology
At the conclusion of the exposure (day 21), all surviving fish were euthanised with an overdose of MS-222 (tricaine methanesulfonate) and separated by sex. Individual total lengths and wet weights were determined. Lengths were measured to the nearest 0.01 mm and wet weights to 0.10 mg. Blood (approximately 20 to 60 μL) was collected from the caudal artery/vein of each fish with a heparinised microhaematocrit capillary tube. Plasma was separated from the blood via centrifugation (approximately 10 minutes at 15 000 g) and stored with protease inhibitors at -75 to -85 °C until analysed for vitellogenin.
Gonads were removed from the euthanised fish by making an incision from the vent forward through the pelvic girdle to the opercula. Gonads were fixed in situ using Davidson's fixative to prevent autolysis and cellular deterioration before removal. For each fish, the gonads were identified by size, shape and colour, and then separated from the other internal organs using stainless steel forceps and scissors. Gonads were transferred to tared tissue cassettes and weighed to the nearest 0.10 mg for the purpose of calculating gonadosomatic index (GSI).
Gonads (right and left) were placed into plastic tissue cassettes which were then placed into an individual container of Davidson's fixative along with the respective fish carcass (volume of fixative equal to at least 10 times the approximated volume of the tissues). After remaining in Davidson's fixative overnight, the tissues were rinsed with reagent grade alcohol and transferred to 10 % neutral buffered formalin in a separate container prior to histopathological analysis.
- Vitellogenin (VTG) Analysis
Plasma samples were analysed for vitellogenin using an enzyme-linked immunosorbent assay (ELISA). The vitellogenin ELISA system kits were manufactured by BioSense Laboratories, Bergen, Norway. Polyconal fathead minnow (Pimephales promelas) VTG antibody and purified VTG protein are utilised in the analysis and provided with the kits. A VTG standard of a separate lot was also purchased separately from the kit and used in the fortification of a culture sample as recommended by OECD (2009).
OBSERVATIONS AND MEASUREMENTS
- Endpoints
A number of endpoints were evaluated over the course of exposure. These endpoints included fecundity (number of eggs per female per reproductive day), fertilisation success, male, female and combined survival, weight (male and female), nuptial tubercle score, blood plasma vitellogenin (VTG) concentration and gonadosomatic index (GSI).
PERFORMANCE CRITERIA
Mean survival of ≥ 90 % of fish in the controls was required over the duration of the exposure and the control fish in each replicate were required to successfully spawn. Evidence of active spawning in the control replicates during the exposure was considered to be achieved if spawning occurred every four days, or if spawning produced approximately 15 eggs per female per day per replicate. During the exposure, greater than 95 % fertility of eggs from the control animals was also required.
Water temperature during the exposure period was also required to differ by no more than 1 °C between test tanks at any one time during the exposure and was required to be maintained within ± 1 °C of the 25 °C temperature. - Details on results:
- EVALUATION OF TEST CONDITIONS
Dissolved oxygen levels ranged from 53 to 110 % of saturation throughout the exposure period. Daily measurements of temperature in the test aquaria and continuous temperature monitoring established that the exposure solution temperatures ranged from 24 to 25 °C in the upper and lower levels during the exposure period. The acceptable performance of the solvent control organisms demonstrated that the dissolved oxygen levels did not adversely impact the performance of the fish in this exposure.
ANALYTICAL RESULTS
The diluter system which prepared and delivered the test solutions to the exposure aquaria functioned properly during the pre-test period and throughout the study. Throughout the study period, all exposure solutions were observed to be clear and colourless. No undissolved test material was observed in the diluter system. Analysis of the exposure solutions during the pre-test period established that the concentrations of test material in the exposure solutions were generally consistent and that the delivery apparatus maintained the expected concentration gradient of the test material.
Since the sampling intervals were not equally spaced (e.g., 5, 14 and 21 days) and exposure concentrations decreased slightly over the course of the exposure, a time-weighted average was calculated (OECD, 1998) for each treatment level. This decrease in concentrations over time was consistent with the observations during previous testing (i.e. the full life cycle test conducted at the testing facility) and was likely due to increased biomass over time and spawning activity.
Time-weighted average concentrations ranged from 85 to 98 % of nominal and defined the treatment levels tested as 0.20, 1.7 and 18 μg a.i./L.
Analysis of 10 of the 12 QC samples resulted in recoveries which were consistent with the predetermined recovery range (i.e., set at 80 to 120 %) and ranged from 80.0 to 114 % of the nominal fortified concentrations (0.100, 2.00 and 20.0 μg a.i./L). Based on these results, it was determined that the appropriate quality control was maintained during the analyses of the test material exposure solution samples. Two of the twelve QC samples were measured at 68.1 and 74.3 % recovery. QC samples can be out of the acceptable range due to a number of factors, some of which are spiking, handling or instrument errors.
BIOLOGICAL OBSERVATIONS
- Survival
Following 21 days of exposure, mean percent survival among male fish exposed to the control and solvent control were 100 and 88 %, respectively (pooled control = 94 %). Percent survival among male fish exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were all 100 %. Statistical analysis (Fisher Exact/Bonferroni-Holm Test) determined no significant difference in percent survival among male fish in any of the treatment levels tested compared to the pooled control (i.e., 94 %).
Percent survival among female fish exposed to the control and solvent control were 88 and 100 %, respectively (pooled control = 94 %). Percent survival among female fish exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were 94, 86 and 86 %, respectively. Statistical analysis (Fisher Exact/Bonferroni-Holm Test) determined no significant difference in percent survival among female fish in any of the treatment levels tested compared to the pooled control (i.e. 94 %).
Percent survival among male and female fish combined exposed to the control and solvent control was 92 and 96 %, respectively (pooled control = 94 %). Percent survival among male and female fish combined exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were 96, 92 and 92 %, respectively. Statistical analysis (Fisher Exact/Bonferroni-Holm Test) determined no significant difference in percent survival among male and female fish combined for any of the treatment levels tested when compared to the pooled control (i.e. 94 %).
- Fertilisation Success and Fecundity
The percentage of viable eggs in the control and solvent control were 96 and 95 %, respectively (pooled control = 95 %). The percentage of viable eggs in the 0.20, 1.7 and 18 μg a.i./L treatment levels were 92, 93 and 96 %, respectively. Statistical analysis (Dunnett’s Test) determined no significant difference in percentage of viable eggs in any of the treatment levels tested compared to the pooled control (i.e. 95 %).
The mean number of eggs per female per reproductive day for the control and solvent control was 10 and 13, respectively (pooled control = 12).The mean number of eggs per female per reproductive day for the 0.20, 1.7 and 18 μg a.i./L treatment levels were 9, 14 and 8, respectively. Statistical analysis (Dunnett’s Test) determined no significant difference in mean number of eggs per female per day among fish exposed to any of the treatment levels tested compared to the pooled control (i.e., 12).
- Male Termination Endpoints
The male weight and GSI of the control and solvent control fish demonstrated that these fish were of similar size and spawning condition.
- Tubercle Score
The mean tubercle score in the control and solvent control were 29 and 37, respectively (pooled control = 33). Mean tubercle scores in the 0.20, 1.7 and 18 μg a.i./L treatment levels were 34, 32 and 28, respectively. Statistical analysis (Jonckheere-Terpstra Step-Down Test) determined no significant difference in mean tubercle scores in the 0.20, 1.7 and 18 μg a.i./L treatment levels compared to the pooled control (i.e. 33).
- Male Weight
The mean male weight in the control and solvent control was 3.1324 and 3.5735 g, respectively (pooled control = 3.3529 g). The mean male weight among fish exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were 3.2101, 3.2910 and 3.0036 g, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in mean male weight in any treatment level tested compared to the pooled control (i.e. 3.3529 g).
- Male GSI
The mean male GSI score in the control and solvent control was 1.60 and 1.64 %, respectively (pooled control = 1.62 %). The mean male GSI score among fish exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were 1.32, 1.57 and 1.52 %, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in mean male GSI score among fish exposed to any of the treatment levels tested compared to the pooled control (i.e., 1.62 %).
- Female Termination Endpoints
The female weight and GSI of the control and solvent control fish demonstrated that these fish were of similar size and spawning condition.
- Female Weight
The mean female weight in the control and solvent control was 1.4164 and 1.5442 g, respectively (pooled control = 1.4803 g). The mean female weight among fish exposed to the 0.20, 1.7 and 18 μg a.i./L treatment levels were 1.6638, 1.5600 and 1.6463 g, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in mean female weight in any treatment levels tested compared to the pooled control (i.e., 1.4803 g).
- Female GSI
The mean female GSI score in the control and solvent control was 13.82 and 11.70 %, respectively (pooled control = 12.76 %). The mean female GSI score in the 0.20, 1.7 and 18 μg a.i./L treatment levels were 10.34, 12.13 and 12.82 %, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in mean female GSI score in any of the treatment levels tested when compared to the pooled control (i.e., 12.76 %).
- Vitellogenin Analysis Results
The mean male vitellogenin concentration in the control and solvent control was 239 and 89 ng/mL, respectively (pooled control = 164 ng/mL). The mean male vitellogenin concentration in the 0.20, 1.7 and 18 μg a.i./L treatment levels were 81, 1375 and 3554 ng/mL, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference for male vitellogenin concentration in any of the treatment levels compared to the pooled control (i.e., 164 ng/mL).
The mean female vitellogenin concentration in the control was 2.0 and 2.7 x 10^6 ng/mL, respectively (pooled control = 2.4 x 10^6 ng/mL). The mean female vitellogenin concentration in the 0.20, 1.7 and 18 μg a.i./L treatment levels were 2.5, 3.0 and 3.2 x 10^6 ng/mL, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in mean female vitellogenin concentration in any of the treatment levels compared to the pooled control (i.e., 2.4 x 10^6 ng/mL).
- Histopathology
H&E-stained tissue sections of ovary and testes were examined from control and treated fish. The only presumptive treatment-related lesion observed was a shift in ovary maturation from stage 3 (stage most commonly observed in controls and group 1 and 2 fish) to stage 2 in group 3 fish. Diagnosis of increased oocyte atresia and testicular degeneration in fish across control and test material treated study groups, as well as concurrent microsporidia infections in many of these cases, suggests the findings were not treatment related. - Conclusions:
- Under the conditions of this study, there were no statistically significant differences observed for any of the endpoints analysed for the test material.
- Executive summary:
The purpose of this test was to assess the potential endocrine activity of the test material in fathead minnow (Pimephales promelas) exposed under flow-through conditions in accordance with the standardised guidelines OECD 229 and US EPA OPPTS 890.1350 under GLP conditions.
The endpoints evaluated were male, female and combined survival, fecundity (number of eggs per female per reproductive day), fertilisation success, nuptial tubercle score, male and female weights, blood plasma vitellogenin (VTG) concentration and gonadosomatic index (GSI).
Pimephales promelas (approximately 24 weeks old) were exposed to the test material in laboratory well water for 21 days at 24 to 25 °C at nominal concentrations of 0.20, 2.0 and 20 μg a.i./L. Time weighted average concentrations were determined to be 0.20, 1.7 and 18 μg a.i./L and the results are based on these.
Under the conditions of this study, there were no statistically significant differences observed for any of the endpoints analysed for the test material.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 June 2011 to 11 July 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA OPPTS 890.1100
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 231
- Deviations:
- no
- Principles of method if other than guideline:
- Although this study was conducted in a frog species, it was considered relevant to include it in this section as part of the overall discussion of endocrine effects.
- GLP compliance:
- yes
- Type of method:
- in vivo
- Endpoint addressed:
- other: Metamorphosis assay; in vivo endocrine system modulation
- Species:
- other: frog
- Strain:
- other: Xenopus laevis
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ORGANISM
Stage 51 X. laevis tadpoles were used to initiate the amphibian metamorphosis assay. The tadpoles originated from adult brood stock maintained at the testing facility. The brood stock was originally obtained from Nasco, Fort Atkinson, Wisconsin and maintained in-house for > 14 days prior to use. The brood stock did not show any sign of sickness, disease, injuries or abnormalities from the day of receipt to the day of pre-exposure initiation. The adult frogs were maintained in a closed-loop re-circulating culture unit. The culture water was FETAX solution; brood stock and test organisms were cultured and tested in FETAX solution from the same source. During the pre-exposure period, the dilution water was characterised as having a dissolved oxygen range of 6.2 to 6.8 mg/L, a pH range of 6.8 to 7.0, a conductivity of 2140 μmhos/cm, a total hardness of 190 mg/L as CaCO₃, and a total alkalinity range of 50 to 52 mg/L as CaCO₃. The area in which the brood stock and test organisms were cultured received a regulated photoperiod of 12 hours of light and 12 hours of darkness. The culture unit was maintained in an isolated, temperature controlled room which maintained water temperature at approximately 22 °C. The isolated room provided a low stress environment for the adult brood frogs and developing tadpoles to be reared and maintained. - Route of administration:
- other: Frogs were exposed in dilution water
- Vehicle:
- acetone
- Details on exposure:
- DILUTION WATER
FETAX solution was used as dilution and control water. Laboratory well water was fortified according to the formulation for FETAX solution presented in "Standard Guide for Conducting the Frog Embryo Teratogenesis Assay – Xenopus (FETAX)" (ASTM, 2004).
Solutions were prepared in 3400-L batches and mixed for approximately 48 hours prior to use to ensure all salts were properly dissolved and that dissolved gasses were at equilibrium with the atmosphere.
Total hardness, total alkalinity, pH and conductivity were monitored on each batch prior to use to ensure that these parameters are within the normal acceptable ranges. The total organic carbon (TOC) of the dilution water was measured on a monthly basis. Total hardness and alkalinity was determined according to Standard Methods for the Examination of Water and Wastewater (APHA, 2005). During the study, weekly characterisation of the FETAX solution established total hardness and alkalinity ranges as CaCO₃ of 72 to 80 mg/L and 140 to 170 mg/L, respectively, a pH range of 8.0 to 8.2, a specific conductance range of 1800 to 2000 μmhos/cm and a TOC range of 0.30 to 0.42 mg/L.
Representative samples of the well water source were analysed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected at concentrations considered toxic in any of the water samples analysed.
Iodide concentrations were also analysed periodically on representative samples of the well water source. For the most recent analysis, iodide concentrations were measured to be approximately 7 μg/L. The acceptable performance of the X. laevis cultures and test organisms, in combination with the previously mentioned analyses, confirmed the acceptability of this dilution water for use during the conduct of bioassays.
STOCK SOLUTION PREPARATION
A glass bead saturator column was used to deliver test material to the exposure system. Columns were constructed using a 40-mm epoxy-coated glass chromatography column. The column was firmly packed with 3 mm Pyrex® glass beads, such that the beads would not loosen during use. A Teflon® filter housing equipped with a 30- to 60-μm Teflon® filter was attached to the output of the column. All fittings used to attach the column to the filter housing and to the water source were composed of Teflon®.
To prepare a column, approximately 1.8 grams of material was diluted with 10 mL of acetone. This solution was poured over the glass beads in the glass column. The column was then attached to a nitrogen gas source to evaporate the acetone. During the evaporation process, the column was rolled to evenly coat the glass surfaces within the column.
Once the solution appeared to be evaporated, the column was detached from the nitrogen source and attached to an FMI pump which delivered a flow of water through the column.
Prior to use, the column was rinsed with dilution water at a flow rate of approximately 120 mL/min for approximately 24 hours. Following this rinsing procedure, the column was set to deliver to the diluter at the appropriate flow rate for dosing.
For this exposure, a saturator column was prepared prior to exposure initiation and on a weekly basis thereafter. Preliminary work demonstrated that a saturator column, prepared in the manner described above, would yield consistent concentration (± 20 % CV) for greater than 7 days. These data are supported by the analytical recoveries for this exposure and preliminary work with this delivery method.
During the preliminary work with this delivery method, the initial flow rate through the saturator column was 120 mL/min. At this flow rate, the saturator column delivered a consistent dose of 100 μg a.i./L. Based on this dose and the target high dose for the exposure, the flow rate through the column was calculated to be 9.9 mL/min. During the pre-exposure period, the saturator column was measured to be delivering approximately 250 μg a.i./L. The reduced flow rate through the saturator column increased the residence time, thereby increasing the dose delivered.
Based on the concentration delivered by the column at this flow rate and the high test concentration, the flow rate through the saturator column was reduced to 4.0 mL/min. At this flow rate, exposure concentrations were measured to be closer to the target dose. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- ANALYTICAL MEASUREMENTS
Prior to the start of the definitive exposure, samples from each replicate of each treatment level and control solution were collected and analysed for test material. In addition, a sample of the diluter stock solution was also analysed. Results of the pre-test analyses were used to judge whether sufficient quantities of test material were being delivered and maintained in the exposure aquaria to initiate the assay.
During the in-life phase of the definitive study, water samples were removed from each treatment level and control on test days 0, 7, 15 and 21. At exposure initiation (day 0), samples were removed from replicate A of each treatment and control solution, and from alternating replicates thereafter. All test samples were removed from the approximate midpoint of each aquarium using a pipette. In addition, a sample of the diluter stock solution was analysed at each sampling interval.
Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. In addition, three QC samples were prepared at each interval and stored frozen for possible analysis. Results of the analyses of the QC samples were used to judge the precision and the quality control maintained during the analysis of exposure solution samples.
All exposure solution and QC samples were analysed for test material by liquid chromatography with mass spectrometry (LC/MS/MS) based on methodology validated at the testing facility. The method validation study established an average recovery of 109 ± 5.03 % from aged FETAX solution. The acceptable range for evaluating QC sample recovery was set at 80 to 120 %.
ANALYTICAL RESULTS
The diluter system which prepared and delivered the test solutions to the exposure aquaria functioned properly during the pre-test period and throughout the definitive study. During the pre-test period, recoveries of test solution and diluter stock solution samples demonstrated that the exposure system was acclimated to test conditions and a concentration gradient was maintained.
Results of the analyses established that the measured concentrations were generally consistent between sampling intervals and that the expected concentration gradient was maintained. The %CV for the high dose (8 μg a.i./L, nominal) slightly exceeded (21 %) guideline criteria for variability (CV≤20 %). This variability is likely attributed to the higher recoveries observed on day 0. If this data set is removed from the calculations, the %CV drops significantly for all doses. When these samples were taken, there was no biomass (animals and feed) within the exposure vessels. It appears that this mass addition impacted exposure concentrations, which resulted in slightly reduced measured concentrations. Mean measured concentrations were approximately 110 % of the nominal levels and defined the treatment levels tested as 0.088, 0.87 and 8.7 μg a.i./L.
Analysis of the QC samples prepared for this study resulted in measured concentrations which were consistent with the predetermined recovery range of 80 to 120 % and ranged from 94.4 to 119 % of the nominal fortified levels (0.0500, 2.00 and 10.0 μg a.i./L). Based on these results, it was demonstrated that satisfactory precision and quality control were maintained during the analysis of exposure solutions.
ANALYTICAL VALIDATION
A method validation was performed to quantify the amount of test material present in aged FETAX solution. Recovery samples were initially diluted with acetonitrile to a final composition of 50:50 acetonitrile:aged FETAX solution (v:v). The mid and high concentration recovery samples were further diluted into the calibration standard range with 50:50 acetonitrile:aged FETAX solution (v:v). All samples were analysed by liquid chromatography/mass spectrometry (LC/MS/MS).
This method validation was conducted by fortification of aged FETAX solution with test material at concentrations of 0.0500, 2.00 and 50.0 μg a.i./L. Recoveries averaged 109 ± 5.03 %, with a limit of quantitation of 0.0182 μg a.i./L. Defined limits for acceptance of quality control sample performance in subsequent studies were set at 80 to 120 %.
- Preparation of Stock Solutions
A 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0562 g of the test material (0.0501 g as active ingredient) in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. Two secondary stock solutions (1.00 and 10.0 mg a.i./L) were prepared by placing 0.0500 and 0.500 mL, respectively, of the 1.00 mg a.i./mL primary stock solution in individual 50.0-mL volumetric flasks and bringing each to volume with acetonitrile. The 1.00 and 10.0 mg a.i./L secondary stock solutions were used to fortify the mid- and high-level recovery samples, respectively. A 10.0 μg a.i./L sub-stock solution was prepared by placing 0.100 mL of the 1.00 mg a.i./L secondary stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution. This 10.0 μg a.i./L sub-stock solution was used to fortify the low-level recovery samples.
An additional 1.00 mg a.i./mL primary stock solution was prepared by placing 0.0562 g of the test material (0.0501 g as active ingredient) in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. A 10.0 mg a.i./L secondary stock solution was prepared by placing 0.500 mL of the 1.00 mg a.i./L primary stock solution in a 50.0-mL volumetric flask and bringing it to volume with acetonitrile. A 100 μg a.i./L sub-stock solution was prepared by placing 0.100 mL of the 10.0 mg a.i./L secondary stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution.
Another sub-stock solution (10.0 μg a.i./L) was prepared by placing 1.00 mL of the 100 μg a.i./L sub-stock solution in a disposable glass bottle and bringing it to a total final volume of 10.0 mL with 50:50 acetonitrile:aged FETAX solution. The 10.0 and 100 μg a.i./L sub-stock solutions were used to prepare calibration standards.
All primary and secondary stock solutions were stored in a refrigerator in glass amber bottles fitted with Teflon®-lined caps. All sub-stock solutions were prepared fresh daily and discarded after use.
- Preparation of Calibration Standards
Calibration standards were prepared in 50:50 acetonitrile:aged FETAX solution using the 10.0 and 100 μg a.i./L sub-stock solutions to yield concentrations of 0.0100, 0.0200, 0.0500, 0.200, 0.500 and 1.00 μg a.i./L.
- Sample Fortification and Preparation
The recovery samples were prepared by fortifying aged FETAX solution with test material to obtain concentrations of 0.0500, 2.00 and 50.0 μg a.i./L. The low-level recovery samples were fortified using the 10.0 μg a.i./L sub stock solution. The mid- and high-level recovery samples were fortified using the 1.00 and 10.0 mg a.i./L secondary stock solutions, respectively. An aliquot (5.00 mL) of each recovery sample were diluted to a final volume of 10 mL with acetonitrile. The mid- and high-level recovery samples (1.00 and 0.0500 mL, respectively) were further diluted into the calibration standard range (to a final volume of 10 mL) with 50:50 acetonitrile:aged FETAX solution (v:v). Three recovery samples were prepared for each concentration. In addition, three recovery samples were left unfortified to serve as controls and were diluted in a similar fashion as the low-level recovery samples.
- Instrumental Conditions
The LC/MS/MS analysis was conducted utilising the following instrumental conditions:
LC parameters:
Column: Phenomenex Synergi MAX-RP, 4 μm, 2.0 mm x 50 mm
Mobile Phase A: 3 mM ammonium acetate in 10:90 acetonitrile:purified reagent water
Mobile Phase B: 3 mM ammonium acetate in 90:10 acetonitrile:purified reagent water
Gradient (flow rate 300 µL/min): At 0.0 and 1.0 minutes 65 % A, 35 % B; at 2.50 and 5.50 minutes 2 % A, 98 % B; and at 7.70 and 9.0 minutes 65 % A, 35 % B
Injection volume: 100 μL
Column oven: 40 °C
Retention Time: approximately 4.3 minutes
MS parameters:
Instrument: MDS Sciex 4000Q TRAP® mass spectrometer equipped with an MDS Sciex 4000Q TRAP® ESI Turbo V Source
Ionisation mode: positive
Q1/Q3 masses: 368.00/231.00 amu
Dwell time: 600 milliseconds
Source temperature: 450 °C
Scan type: MRM
- Preparation of Standard Curve
Two sets of calibration standards were analysed with each recovery sample set; one set prior to analysis of the recovery samples, and the second set immediately following the analysis of the recovery samples. Injection of samples and calibration standards onto the LC/MS/MS system was performed by programmed automated injection.
- Calculations
A calibration curve was constructed by plotting the analyte concentration (μg a.i./L) in the calibration standards against the peak area of the calibration standards. The equation of the line (equation 1) was algebraically manipulated to give equation 2. The concentration of the test material within each recovery sample was determined using the regression coefficients from the quadratic equation, the peak area of the recovery sample, and the dilution factor. Equations 2 and 3 were then used to calculate measured concentrations and analytical results.
Equation 1: y = ax² + bx +c
Equation2: DC (x) = (-b + √(b² - 4aC)) / 2a
Equation 3: A DC x DF
where:
y = detector response (peak area) for analyte
a, b and c = regression constants
DC (x) = detected concentration (μg a.i./L) in the sample
C = constant c minus the peak area; C = (c - y)
DF = dilution factor (the final sample volume divided by the original sample volume)
A = concentration of the analyte in the original sample
The limit of quantitation (LOQ) was calculated using the following:
Area(MIN) = A(LS)
LOQ(inst) = -b + √(b² - 4aC)
LOQ = LOQ(inst) x DF(ctrl)
where:
AreaMIN = mean detector response (peak area) of the low concentration calibration standard (two injections)
ALS = mean detector response (peak area) of the low concentration calibration standard (two injections)
a, b, c = regression constants
C = regression constant c – Area(MIN); C = (c - Area(MIN))
LOQ(inst) = limit of quantitation on the instrument
DF(ctrl) = dilution factor of the control samples (smallest dilution factor used)
LOQ = limit of quantitation reported for the analysis - Duration of treatment / exposure:
- 21 days
- Frequency of treatment:
- continuous in dilution water
- Dose / conc.:
- 0.08 other: μg a.i./L (nominal)
- Dose / conc.:
- 0.8 other: μg a.i./L (nominal)
- Dose / conc.:
- 8 other: μg a.i./L (nominal)
- No. of animals per sex per dose:
- All test vessels contained 20 tadpoles.
- Control animals:
- yes
- Details on study design:
- An amphibian metamorphosis assay with the African clawed frog (Xenopus laevis) was carried out. The endpoints evaluated were developmental stage, snout-vent length, hind limb length, hind limb length (normalised), whole body wet weight and thyroid gland histology.
TEST CONCENTRATIONS
Selection of nominal test material concentrations for the assay was based on a preliminary 96-hour acute exposure performed at the testing facility; nominal test material concentrations of 0.080, 0.80 and 8.0 μg a.i./L were selected for the definitive exposure. This dose range utilises 1/3 of the 96-hour acute LC50 as the high dose and a dilution factor of 10. Concentrations were adjusted for the purity of the test material and are presented as active ingredient (a.i.).
PRELIMINARY ACUTE EXPOSURE
After an initial preliminary exposure in which significant mortality was seen, the range-finding exposure was initiated at nominal concentrations of 2.3, 4.7, 9.4, 19, 38 and 75 μg/L.
The acute exposure was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature controlled water bath, and a set of fourteen exposure aquaria with a total solution volume of 6.5 L. The exposure was initiated with stage 51 X. laevis tadpoles. Two replicates were maintained for each concentration and control.
Each replicate vessel contained 10 tadpoles at exposure initiation. Test organisms were monitored daily for observations of mortality and abnormal behaviour. During the exposure, dissolved oxygen, pH and temperature were monitored daily. At exposure initiation, samples were taken from one replicate of each concentration and control to determine actual exposure concentrations.
EXPOSURE SYSTEM
Prior to exposure initiation, an FMI pump was calibrated to deliver 4.0 mL/min (36 mL/cycle) of the diluter stock solution (250 μg a.i./L) into the diluter's chemical mixing chamber, which received 1.075 L of dilution water per cycle, for a total of 1.11 L per cycle. The mixing chamber was positioned over a magnetic stir plate which aided in mixing the stock solution with the dilution water. The solution contained in the mixing chamber constituted the highest nominal test concentration (8.0 μg a.i./L) and was subsequently proportionally diluted (10 %) to provide the remaining nominal exposure concentrations (0.80 and 0.080 μg a.i./L).
A set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels and contained no test material.
The metamorphosis assay was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath and a set of 16 exposure aquaria. The exposure system was designed to provide three concentrations of the test material, and a dilution water control to four replicate test aquaria. The exposure system was constructed of glass, silicone sealant, stainless steel, and Teflon®. Each 2.5-gallon exposure aquaria measured 30 x 14.5 x 20 cm with a 14.5-cm high side drain that maintained a constant exposure solution volume of approximately 6.5 L. Flow-splitting cells were employed to equally distribute the solutions to the four replicate vessels for each concentration or control group at a rate of 250 mL of test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within ± 5 % of the nominal value. The exposure system was operating properly for nine days prior to exposure initiation to allow equilibration of the test material in the diluter apparatus and exposure aquaria. All test chambers were brushed and siphoned daily to remove detritus and uneaten food during the test.
During the study, the diluter delivered the control and test solutions to the exposure aquaria at a rate sufficient to provide approximately 6.2 aquarium volumes per 24-hour period, with a 90 % replacement time of approximately 9 hours. Calibration of the diluter system was conducted prior to exposure initiation and confirmed at test termination by measuring delivery volumes of toxicant and dilution water. The function of the diluter was monitored daily and a visual check was performed twice daily.
In addition, weekly analysis of exposure solutions for test material concentration was also used to verify proper operation of the diluter system.
TEST CONDITIONS
The exposure was conducted in a water bath containing circulating water designed to maintain the test solution temperatures at 22 ± 1 °C. Two Process Technology USA Immersion heaters with an Omega CN 76000 Series temperature controller were used to regulate temperatures. Total dissolved oxygen concentration was maintained at > 40 % of air saturation (3.5 mg/L). The pH of the dilution water was maintained at 6.5 to 8.5, with inter-replicate and inter-treatment differentials < 0.5. Illumination was provided by Sylvania Oktron® fluorescent bulbs centrally located above the test aquaria. A photoperiod of twelve hours of light at 56 to 110 footcandles (600 to 1200 lux) and 12 hours of darkness at the exposure solution surface were provided each day using an automatic timer. The exposure system was curtained off to avoid disruption by the standard 16:8 laboratory photoperiod.
PRE-EXPOSURE PERIOD
On pre-exposure day 0, three pairs of adult male and female X. laevis were induced to breed. Each frog was provided a primary injection of human chorionic gonadotropin (hCG) in the morning (0.25 cc for both males and females) and a secondary injection in the afternoon (0.25 cc for males and 0.75 cc for females). Following these injections, each pair was placed into a separate 21-L breeding aquarium. Each breeding aquarium contained a false bottom, which allowed the egg masses to fall to the bottom of the aquarium and remain there unharmed. The following morning (pre-exposure day 1), the adult frogs and the false bottoms were removed from the breeding aquaria. The embryos remained in their respective breeding aquarium until they hatched and reached feeding stage (approximately 5 days post-fertilisation). During this time, the quality of each of the three spawns was quantitatively assessed; criteria were embryo survival and the quantity of embryos.
Once all tadpoles were at feeding stage (day 5 post fertilisation), larvae from the highest quality spawn were transferred to nine, 10-L rearing tanks; all tadpoles originated from a single spawn. One-hundred tadpoles were transferred to each rearing tank for a larval density of 10 tadpoles/L.
Additional larvae were maintained in the original breeding tank to be used as replacements in the event that mortalities occur in the rearing tanks during the first week. This procedure maintained consistent organism density and thereby reduced developmental divergence during the pre-exposure period.
During this pre-exposure period, tadpoles were maintained under flow-through conditions similar to that of the actual exposure. Flow rates and temperature were monitored daily; dissolved oxygen and pH were measured on pre-exposure days 5 and 8. Temperature was also monitored continuously in a representative rearing tank and recorded daily. During this period, flow rates were maintained at approximately 50 mL/min, temperatures ranged from 21 to 25 °C, dissolved oxygen ranged from 6.1 to 7.5 mg/L, and pH ranged from 6.8 to 7.4.
Tadpoles were evaluated daily for mortalities, proper development and overall health. On pre-exposure days 9 and 12, representative tadpoles of the pre-exposure population were inspected for developmental stage in order to estimate the appropriate time point for initiation of exposure.
Tadpole rearing tanks were siphoned clean daily during this period. Care was taken to minimise stress on the tadpoles during inspection and cleaning of aquaria.
FEEDING
During the pre-exposure and in-life exposure periods, tadpoles were fed Xenopus Express Tadpole Food (Xenopus Express, Brooksville, Florida). Xenopus Express Tadpole Food is a fine particulate that stays suspended in the water column for a long period of time and hence, it is subject to washing out with the flow. The Tadpole Food was fed as a stock solution prepared in dilution water. The stock was prepared according to supplier specifications and was refrigerated when not in use.
During the pre-exposure period, tadpoles were fed twice per day. The daily food ration was 5, 10, 30 and 30 mg/tadpole on days 5 to 6, 7 to 10, 11 to 14 and 15 to 19, respectively.
For the in-life portion of the assay, the daily food ration was 30, 40, 50, 70 and 80 mg/tadpole on days 0 to 4, 5 to 7, 8 to 10, 11 to 14 and 15 to 21, respectively.
EXPOSURE INITIATION
On pre-exposure day 12 (day 12 post-fertilisation) a sufficient number of tadpoles reached stage 51, therefore the exposure initiation process began. All tadpoles from each rearing vessel were removed and pooled into a vessel containing dilution water maintained at approximately 22 °C. For selection, normal, healthy tadpoles were removed from the pooled group, transferred to a deep well Petri dish and observed under a binocular dissection microscope to determine the developmental stage. Staging was conducted on each individual tadpole and as accurately as possible. The primary developmental landmark for staging stage 51 tadpoles was hind limb morphology.
Tadpoles that met the staging criteria were held in a container of control water at 22 ± 1 °C until the staging process was complete. Tadpoles that did not meet these criteria were placed in a separate container. Once the staging process was complete, the larvae were transferred into the randomly assigned test vessels. Tadpoles were distributed to the test vessels four at a time until each vessel contained four tadpoles. This process was repeated until all test vessels contained 20 tadpoles. Following distribution, each test vessel was examined for tadpoles exhibiting abnormal behaviour and appearance. At this time, abnormal tadpoles were removed from the respective test vessel and replaced with healthy tadpoles.
WATER QUALITY MEASUREMENTS
Dissolved oxygen concentration, pH and temperature measurements were taken in all vessels on day 0 and in alternating replicates daily thereafter. Total hardness, total alkalinity and specific conductance were measured in replicate A of the control, low and high test concentrations on day 0 and in alternating replicates weekly thereafter. Test solution temperature was continuously monitored in replicate B of the control. - Examinations:
- BIOLOGICAL METHODS
- Day 7 Measurements
On day 7, five tadpoles were randomly selected from each test vessel for growth metrics. For this procedure, a unique set of random numbers were generated in the range of 1 to 20 for each replicate concentration. The first 5 numbers generated for a set would represent the tadpole to be selected for growth metrics. For each replicate vessel, all tadpoles were removed and placed into a secondary container containing dilution water at the appropriate temperature. Each tadpole was then removed from the secondary container and counted in ascending order. When the appropriate random number was reached, that tadpole would be selected for termination. If there were not a sufficient number of tadpoles within a respective tank for the corresponding number (e.g., #20 generated, but only 19 tadpoles are in the vessel), the number would be skipped and the next number (e.g., the 6th number generated) would be used until a total of five tadpoles were selected.
Tadpoles selected for measurement were euthanised in a vessel containing dilution water and MS-222. Tadpoles not selected for measurement were returned to their respective exposure vessels.
Euthanised tadpoles were then removed from the euthanising solution and digital images were taken of each tadpole for snout-vent length and hind limb length. Developmental stage was then determined for each tadpole using a binocular dissection microscope. Each tadpole was then blotted dry prior to body weight determination to the nearest 0.1 mg. Digital photographs were taken of each tadpole for snout-vent length and hind limb length measurements. Images were obtained using the following hardware and software: Zeiss Stemi-2000 CS, a Zeiss AxioCam ICc 3 and Zeiss AxioVision image analysing software.
- Day 21 Measurements
At test termination (day 21), the remaining tadpoles were removed from the test vessels and euthanised in a vessel containing dilution water and MS-222. Tadpoles were then removed from the euthanising solution and digital images were taken of each tadpole for snout-vent length and hind limb length measurements using the equipment and procedures described above.
Developmental stage was then determined for each tadpole using a binocular dissection microscope. Each tadpole was then blotted dry prior and weighed to the nearest 0.1 mg.
Following these procedures, each tadpole was transferred to individually labelled 60-mL Nalgene® containers. Each container was then filled with approximately 30 mL of Davidson’s fixative. Tadpoles remained submerged, as whole body samples, in Davidson’s fixative for approximately 72 hours. Samples were then rinsed with 70 % reagent grade ethanol and stored in 30 mL of 10 % neutral buffered formalin.
BIOLOGICAL ENDPOINTS
- Developmental Stage
Developmental stage was determined at days 7 and 21. Developmental stage data were used to determine if development was accelerated, asynchronous, delayed, or unaffected.
- Snout-Vent Length
Snout-vent length was determined at days 7 and 21. Snout-vent length was measured for each individual tadpole to the nearest 0.01 mm. For consistency, the cranial aspect of the vent was used as the caudal limit of the measurement.
- Hind Limb Length
Hind limb length was determined at days 7 and 21. For consistency, the hind limb length is measured on the left hind limb. On day 7, hind limb measurement is straight forward. On day 21, measuring hind limb length is more complicated due to natural bends in the limb. Therefore, measurements of hind limb length originated at the body wall and followed the midline of the limb through any angular deviations. Hind limb length is expressed as the total length, as described above, and as a normalised value. Hind limb length, normalised by snout-vent length, was calculated by taking the ratio of the hind limb length to snout-vent length of each individual.
- Whole Body Wet Weight
Whole body wet weight was determined at days 7 and 21. Body weights were measured on an analytical balance to the nearest 0.1 mg for each individual tadpole.
- Thyroid Gland Histology
For histological analyses, a total of five tadpoles were selected from each replicate test concentration. For tadpole selection, a unique set of random numbers were generated in the range of 6 to 20 for each replicate concentration. Each number represented the tadpole identification number for the respective replicate concentration. Tadpoles were selected in order (top to bottom) based on the numbers generated and the following criteria. Tadpoles selected for histological analysis were stage-matched to the median of the control (stage 60), if possible. If there were replicates with more than five larvae at the appropriate stage, then five larvae were randomly selected. If there was not a sufficient number of tadpoles at the appropriate stage, the next stage down was selected (stage 59), until a total of five tadpoles were selected. If an insufficient number of tadpoles still remained, the upper stage from the median of the control was selected until a total of five tadpoles were selected.
- Additional Observations
All test vessels were examined daily for survival and behavioural assessment. Dead animals were removed from the test tank and recorded when observed. Observations of abnormal behaviour, differences in food consumption, grossly visible malformations or lesions were also recorded. - Details on results:
- PRELIMINARY TESTING
In the preliminary test, the 96 hour LC50 was determined to be 24 µg a.i./L with a confidence interval of 21 to 27 μg a.i./L, calculated using the Spearman-Karber method.
DEFINITIVE TESTING
- Evaluation of Test Conditions
During the study, daily measurements of the test solutions established a dissolved oxygen concentration range of 4.0 to 8.4 mg/L (45 to 97 % of saturation). Continuous temperature monitoring demonstrated that the temperature ranged from 21 to 23 °C throughout the exposure; inter-replicate and inter-treatment differentials did not exceed 0.5 °C.
Daily temperature monitoring established a range of 22 to 23 °C. Measurements of pH ranged from 7.3 to 8.1; inter-replicate and inter-treatment differentials did not exceed 0.5. Weekly characterisation of the high, low and control solutions established total hardness and alkalinity ranges as CaCO₃ of 110 to 160 mg/L and 50 to 78 mg/L, respectively, and a specific conductance range of 950 to 1800 μmhos/cm.
The water quality parameters measured were not affected by the established concentration gradient of the test material. The measurements established that the water quality parameters maintained throughout the 21-day exposure remained within an acceptable range for the promotion of tadpole survival and growth.
BIOLOGICAL ENDPOINTS
A summary of the 7- and 21-day biological endpoints measured are presented in Tables 1 and 2, respectively.
- Mortality and Biological Observations
Following 21 days of exposure, one mortality was observed in the 0.088 μg a.i./L treatment level and two mortalities each were observed in the 0.87 and 8.7 μg a.i./L treatment levels. No mortality was observed in the control. Beginning on test day 10, several tadpoles exposed to the control and 0.088 μg a.i./L treatment level were observed to be deformed (e.g. spinal curvature), while tadpoles exposed to the 0.87 μg a.i./L treatment level exhibited this deformity beginning on test day 15. Tadpoles exposed to the 8.7 μg a.i./L treatment level were observed to be deformed (e.g. spinal curvature) beginning on test day 19.
- Whole Body Wet Weight
The mean day 7 wet weight for tadpoles exposed to the control averaged 0.3579 g. Day 7 wet weight for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 0.4134, 0.4099 and 0.3504 g, respectively. Dunnett's Multiple Comparison Test determined a significant increase in day 7 whole body wet weight among tadpoles exposed to the 0.088 and 0.87 μg a.i./L treatment levels compared to the control.
The mean day 21 wet weight for tadpoles exposed to the control averaged 0.9899 g. Day 21 wet weight for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 0.9837, 0.9820 and 1.0518 g, respectively. Dunnett's Multiple Comparison Test determined no significant difference in day 21 whole body wet weight among tadpoles exposed to any of the treatment levels tested compared to the control.
- Snout-Vent Length (SVL)
The mean day 7 snout-vent length for tadpoles exposed to the control averaged 18.45 mm. Day 7 snout-vent length for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 19.33, 19.18 and 18.02 mm, respectively. Dunnett's Multiple Comparison Test determined a significant increase in day 7 snout-vent length among tadpoles exposed to the 0.088 and 0.87 μg a.i./L treatment levels compared to the control.
The mean day 21 snout-vent length for tadpoles exposed to the control averaged 24.69 mm. Day 21 snout-vent length for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 24.53, 24.39 and 25.13 mm, respectively. Dunnett's Multiple Comparison Test determined no significant difference in day 21 snout-vent length among tadpoles exposed to any of the treatment levels tested compared to the control.
- Hind Limb Length
The mean day 7 hind limb length for tadpoles exposed to the control averaged 2.65 mm. Day 7 hind limb length for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 2.79, 2.70 and 2.47 mm, respectively. Dunnett's Multiple Comparison Test determined no significant difference in day 7 hind limb length among tadpoles exposed to any of the treatment levels tested compared to the control.
The mean day 21 hind limb length for tadpoles exposed to the control averaged 17.46 mm. Day 21 hind limb length for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 17.93, 18.07 and 16.56 mm, respectively. Dunnett's Multiple Comparison Test determined no significant difference in day 21 hind limb length among tadpoles exposed to any of the treatment levels tested compared to the control.
- Hind Limb Length Normalised by Snout-Vent Length
The mean day 7 hind limb length, normalised by SVL, for tadpoles exposed to the control averaged 0.14 mm. Day 7 hind limb length, normalised by SVL, for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 0.14 mm. Dunnett's Multiple Comparison Test determined no significant difference in day 7 hind limb length, normalised by SVL, among tadpoles exposed to any of the treatment levels tested compared to the control.
The mean day 21 hind limb length, normalised by SVL, for tadpoles exposed to the control averaged 0.73 mm. Day 21 hind limb length, normalised by SVL, for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels averaged 0.75, 0.76 and 0.67 mm, respectively.
Dunnett's Multiple Comparison Test determined no significant difference in day 21 hind limb length, normalised by SVL, among tadpoles exposed to any of the treatment levels tested compared to the control.
- Developmental Stage
The median day 7 developmental stage for tadpoles exposed to the control averaged 54. The median day 7 developmental stage for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels all averaged 54. Jonckheere-Terpstra Step-Down Test determined no significant difference in day 7 developmental stage among tadpoles exposed to any of the treatment levels tested compared to the control.
The median day 21 developmental stage for tadpoles exposed to the control averaged 60. The median day 21 developmental stage for tadpoles exposed to the 0.088, 0.87 and 8.7 μg a.i./L treatment levels all averaged 60. Jonckheere-Terpstra Step-Down Test determined no significant difference in day 21 developmental stage among tadpoles exposed to any of the treatment levels tested compared to the control.
- Thyroid Histology
All tissues submitted from 21-day assay were examined via histopathology. Test material related findings included markedly increased incidence of mild follicular cell hypertrophy; increased incidence of follicular cell hyperplasia with increasing dose concentration; and colloid depletion/changes in colloid quality in the high dose group (8.0 μg a.i./L, nominal). - Conclusions:
- The test material was found to significantly increase both 7-day wet weight and 7-day snout-vent length at 0.088 and 0.87 μg a.i./L. The test material was not found to significantly increase or decrease any other apical endpoint. Asynchronous development or significant acceleration or delay of median Nieuwkoop-Faber (NF) developmental stage was not observed at 7 days or 21 days for any treatment level. Effects on thyroid gland histopathology were observed at 0.87 μg a.i./L.
- Executive summary:
A metamorphosis assay was conducted in accordance with the standardised guidelines US EPA OPPTS 890.1100 and OECD 231 under GLP conditions.
The 21-day assay of the test material on amphibian metamorphosis of the African clawed frog (Xenopus laevis) was studied under flow-through conditions. Tadpoles, stage 51 and 12 days old post fertilisation at exposure initiation were exposed to the test material at concentrations of 0.080, 0.80 and 8.0 μg a.i./L. the study was conducted at 21 to 23 °C using FETAX solution as dilution water.
The test material was found to significantly increase both 7-day wet weight and 7-day snout-vent length at 0.088 and 0.87 μg a.i./L. The test material was not found to significantly increase or decrease any other apical endpoint. Asynchronous development or significant acceleration or delay of median Nieuwkoop-Faber (NF) developmental stage was not observed at 7 days or 21 days for any treatment level. Effects on thyroid gland histopathology were observed at 0.87 μg a.i./L. Histopathological effects are an increased incidence of mild follicular cell hyperplasia with increased dose concentration; a marked increase in the incidence of mild follicular cell hypertrophy in dose group 1; and mild colloid depletion and changes in the colloid quality at 8.7 μg a.i./L. Eighty-five percent of animals at 8.7 μg a.i./L showed mild follicular cell hypertrophy, a rate much higher than that observed at the control (21 % of animals) and at 0.088 and 0.87 μg a.i./L (35 % of animals), suggesting a dosing related effect. Likewise, a dose related response was indicated by the increased incidence of mild follicular cell hyperplasia with increased dose concentration: 11 % at the control; 40 % at 0.088 μg a.i./L; 50 % at 0.87 μg a.i./L; and 80 % at 8.7 μg a.i./L. While the hypertrophy/hyperplasia observed in all study groups were mild (Grade 1 affecting 30-50 % of follicles) the changes in the higher dose animals tended towards the higher end of the grading scale. In two animals (1A10 and 2C10) with follicular cell hypertrophy and hyperplasia there also was mild thyroid gland hypertrophy, but in most cases there was not an overall increase in glandular size. In twenty-eight animals, the follicular hypertrophy and hyperplasia occurred concurrently, which is consistent with rapid cell growth / proliferation. Additional findings observed predominately at 8.7 μg a.i./L include mild colloid depletion in 25 % of animals and changes in colloid quality with foamy or more rarely granular colloid present in 50 % of animals.
These colloid changes at 8.7 μg a.i./L occurred concurrently with follicular cell hypertrophy and/or hyperplasia. All effects are reported based on comparison to the negative (clean water) control.
Referenceopen allclose all
Table 1: Androgen Agonist- Body Weight Changes
Dose Group |
Dose Level (mg/kg/day) |
n |
Initial Mean BW (g) ± SD |
Final Mean BW (g) ± SD |
Final BW (% of Control) |
Mean BW Changes (g) ± SD |
Vehicle Control (corn oil) |
0 |
8 |
241.9 ± 16.1 |
292.9 ± 24.9 |
- |
51.0 ± 10.1 |
Test Material |
47 |
8 |
243.9 ± 13.5 |
281.4 ± 25.1 |
96.1 |
37.5 ± 16.0
|
Test Material |
150 |
8 |
235.9 ± 14.1 |
248.7 ± 22.9* |
84.9 |
12.9 ± 24.1* |
TP (Positive Control) |
0.4 |
8 |
241.3 ± 10.2 |
311.6 ± 21.4 |
- |
70.3 ± 12.7† |
BW: Body weight
TP: Testosterone propionate
*Statistically significant (p<0.05) compared to the vehicle control mean (Dunnett’s test)
†Statistically significant (p<0.05) compared to the vehicle control mean (t-test)
Table 2: Androgen Antagonist- Body Weight Changes
Dose Group |
Dose Level (mg/kg/day) |
n |
Initial Mean BW (g) ± SD |
Final Mean BW (g) ± SD |
Final BW (% of Control)
|
Mean BW Changes (g) ± SD |
TP (Negative Control) |
0.4 |
8 |
241.3 ± 10.2 |
311.6 ± 21.4 |
- |
70.3 ± 12.7 |
Test Material + TP |
15 + 0.4 |
8 |
243.0 ± 16.3 |
305.0 ± 24.8 |
97.9 |
62.0 ± 12.4 |
Test Material + TP |
47 + 0.4 |
8 |
240.2 ± 12.2 |
292.5 ± 15.5 |
93.9 |
52.3 ± 6.5* |
Test Material + TP |
150 + 0.4 |
8 |
241.0 ± 10.0 |
257.3 ± 19.7* |
82.6 |
16.3 ± 20.8* |
FT + TP (Positive Control) |
3.0 + 0.4 |
8 |
240.3 ± 16.5 |
302.1 ± 27.8 |
- |
61.8 ± 12.9 |
BW: Body weight
TP: Testosterone propionate
FT: Flutamide
*Statistically significant (p<0.05) compared to the negative control mean (Dunnett’s test)
Table 3: Androgen Agonist; Androgen Dependent Tissue Weights
Dose Group |
Dose Level (mg/kg/day) |
n |
Mean Glans Penis Weight (mg) ± SD (CV) |
Mean Cowper’s Glands Weight (mg) ± SD (CV) |
Mean LABC Weight¹ (mg) ± SD (CV) |
Mean Ventral Prostate Weight (mg) ± SD (CV) |
Mean Seminal Vesicles Weight (mg) ± SD (CV) |
Vehicle Control (corn oil) |
0 |
8 |
38.6 ± 14.8 (38) [22] |
4.6 ± 3.3 (71) [55] |
136.0 ± 19.1 (14) |
10.0 ± 4.8 (48) [45] |
26.8 ± 5.3 (20) |
Test Material |
47 |
8 |
36.6 ± 9.4 (26) |
2.8 ± 1.4 (50) |
125.7 ± 32.2 (26) |
12.9 ± 6.9 (54) |
23.5 ± 5.8 (24) |
Test Material |
150 |
7 |
37.2 ± 6.1 (16) |
4.0 ± 3.3 (83) [55] |
105.9 ± 16.6 (16) |
9.6 ± 3.9 (40) |
25.8 ± 6.5 (25) |
TP (Positive Control) |
0.4 |
7 |
81.7 ± 14.3† (17) |
34.0 ± 2.0† (6) |
526.7 ± 56.2† (11) |
163.1 ± 30.9† (19) |
476.4 ± 100.6† (21) |
LABC: levator ani plus bulbocavernous muscle complex
TP: Testosterone propionate
[ ]Indicate maximum acceptable CV for mean tissue weight; all other CV values are below the maximum allowable CV
*Statistically significant (p<0.05) compared to the vehicle control mean (Dunnett’s test)
†Statistically significant (p<0.05) compared to the vehicle control mean (t-test)
¹ANOVA p value was between 0.05 and 0.1
Table 4: Androgen Antagonist; Androgen Dependent Tissue Weights
Dose Group |
Dose Level (mg/kg/day) |
n |
Mean Glans Penis Weight (mg) ± SD (CV) |
Mean Cowper’s Glands Weight (mg) ± SD (CV) |
Mean LABC Weight¹ (mg) ± SD (CV) |
Mean Ventral Prostate Weight (mg) ± SD (CV) |
Mean Seminal Vesicles Weight (mg) ± SD (CV) |
TP (Negative Control) |
0.4 |
7 |
81.7 ± 14.3 (17) |
34.0 ± 2.0 (6) |
526.7 ± 56.2 (11) |
163.1 ± 30.9 (19) |
476.4 ± 100.6 (21) |
Test Material + TP |
15 + 0.4 |
8 |
72.8 ± 11.3 (15) |
32.6 ± 7.3 (23) |
444.3 ± 82.1* (18) |
133.8 ± 25.1* (19) |
443.5 ± 83.2 (19) |
Test Material + TP |
47 + 0.4 |
8 |
75.2 ± 13.5 (18) |
26.9 ± 2.4 (9) |
383.5 ± 67.3* (18) |
121.3 ± 23.0* (19) |
428.8 ± 82.5 (19) |
Test Material + TP |
150 + 0.4 |
8 |
74.4 ± 7.3 (10) |
29.2 ± 7.7 (26) |
330.1 ± 38.4* (12) |
96.9 ± 11.4* (12) |
318.3 ± 55.1* (17) |
FT + TP (Positive Control) |
3.0 + 0.4 |
8 |
55.6 ± 9.7† (17) |
11.1 ± 3.5† (32) |
221.4 ± 49.0† (22) |
30.2 ± 17.2† (57) |
67.3 ± 17.2† (26) |
LABC: levator ani plus bulbocavernous muscle complex
TP: Testosterone propionate
*Statistically significant (p<0.05) compared to the negative control mean (Dunnett’s test)
†Statistically significant (p<0.05) compared to the negative control mean (t-test)
Table 1: Number of Ovary Follicles, Follicular Cysts and Corpora Lutea in Female Rats
Effect |
Dose Group |
|||||||||||
Control |
62.5 mg/kg/day |
125 mg/kg/day |
||||||||||
Mean |
SD |
CV |
n |
Mean |
SD |
CV |
n |
Mean |
SD |
CV |
n |
|
Small Follicles |
22.4 |
7.2 |
32.1 |
16 |
28.4 |
11.6 |
40.8 |
16 |
33.9* |
14.5* |
42.8* |
14* |
Medium Follicles |
5.6 |
2.8 |
50.0 |
16 |
6.0 |
1.7 |
28.3 |
16 |
7.2 |
3.0 |
41.7 |
14 |
Antral Follicles |
9.1 |
4.5 |
49.5 |
16 |
10.3 |
4.4 |
42.7 |
16 |
12.0 |
3.2 |
26.7 |
14 |
Antretic Follicles |
8.6 |
2.8 |
32.6 |
16 |
9.0 |
3.8 |
42.2 |
16 |
8.2 |
2.2 |
26.8 |
14 |
Follicular Cysts |
0.1 |
0.2 |
200.0 |
16 |
0.1 |
0.2 |
200.0 |
16 |
0.7* |
0.9 |
128.6* |
14* |
Corpora Lutea |
5.9 |
1.5 |
25.4 |
16 |
5.0 |
2.0 |
40 |
16 |
2.2* |
1.9 |
8.3* |
13* |
The pairwise test used was Dunnett’s test, with the exception of the follicular cysts endpoint for which Dunn’s test was used.
*Statistically different compared to the vehicle control mean
Table 2: Female Thyroid Incidence: Follicular Cell Height and Colloid Area
|
Dose Group |
||
Control |
62.5 mg/kg/day |
125 mg/kg/day |
|
n |
16 |
16 |
14 |
Follicular cell (height) |
|||
1 |
6 (37.5 %) |
1 (6.3 %) |
1 (7.1 %) |
2 |
10 (62.5 %) |
14 (87.5 %) |
7 (50.0 %) |
3 |
0 |
1 (6.3 %) |
6 (42.9 %) |
4 |
0 |
0 |
0 |
5 |
0 |
0 |
0 |
Follicular colloid (area) |
|||
1 |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
3 |
0 |
1 (6.3 %) |
5 (35.7 %) |
4 |
6 (37.5 %) |
13 (81.3 %) |
8 (57.1 %) |
5 |
10 (62.5 %) |
2 (12.5 %) |
1 (7.1 %) |
Scale: A five-point grading scale (1 = shortest / smallest; 5 = tallest / largest) was used and the thyroids were evaluated relative to photomicrographs supplied by the US EPA.
Table 3: Male Thyroid Incidence: Follicular Cell Height and Colloid Area
|
Dose Group |
||
Control |
62.5 mg/kg/day |
125 mg/kg/day |
|
n |
16 |
16 |
16 |
Follicular cell (height) |
|||
1 |
1 (6.3 %) |
0 |
0 |
2 |
8 (50.0 %) |
2 (12.5 %) |
1 (6.3 %) |
3 |
6 (37.5 %) |
9 (56.3 %) |
12 (75.0 %) |
4 |
1 (6.3 %) |
5 (31.3 %) |
3 (18.8 %) |
5 |
0 |
0 |
0 |
Follicular colloid (area) |
|||
1 |
0 |
0 |
0 |
2 |
1 (6.3 %) |
5 (31.3 %) |
3 (18.8 %) |
3 |
4 (25.0 %) |
9 (56.3 %) |
12 (75.0 %) |
4 |
9 (56.3 %) |
2 (12.5 %) |
1 (6.3 %) |
5 |
2 (12.5 %) |
0 |
0 |
Scale: A five-point grading scale (1 = shortest / smallest; 5 = tallest / largest) was used and the thyroids were evaluated relative to photomicrographs supplied by the US EPA.
Table 1: Group Mean Initial, Final, and Body Weight Changes
Dose Group |
Dose Level (mg/kg/day) |
n |
Initial Mean Body Weight (g) ± SD |
Final Mean Body Weight (g) ± SD |
Mean Body Weight Change (g) ± SD |
Vehicle Control |
0 |
8 |
247.9 ± 20.8 |
262.3 ± 21.9 |
14.4 ± 3.4 |
Test Material |
125 |
8 |
250.0 ± 19.5 |
261.5 ± 21.9 |
11.5 ± 4.9 |
Test Material |
400 |
8 |
246.3 ± 14.1 |
230.0 ± 25.6* |
-16.3 ± 14.3* |
*Statistically significant (p<0.05) compared to the vehicle control (Dunnett’s test)
Table 2: Absolute Uterine Weights of Female Rats
Dose Group |
Dose Level (mg/kg/day) |
n |
Mean Uterine Weight- Wet (mg) ± SD |
Mean Uterine Weight- Blotted (mg) ± SD |
Vehicle Control |
0 |
8 |
58.6 ± 12.1 |
52.5 ± 9.5 |
Test Material |
125 |
8 |
61.5 ± 6.5 |
54.4 ± 7.6 |
Test Material |
400 |
8 |
71.4 ± 14.3 |
63.6 ± 11.1 |
17α-ethinyl estradiol |
0.1 |
8 |
154.6 ± 98.7* |
118.5 ± 34.5* |
*Statistically significant (p<0.05) compared to the vehicle control (t-test)
Table 1: Results of MTT Cell Viability Assay (percentage of solvent control)
Condition / Dose (µM) |
Cell Viability - Run 2 |
Cell Viability - Run 3 |
Cell Viability - Run 4 |
|||
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
QC Plate |
||||||
Blank |
104.3 |
2.69 |
104.3 |
2.81 |
100.7 |
3.98 |
Background |
95.4 |
4.14 |
102.5 |
1.79 |
98.9 |
0.94 |
SC + Methanol |
5.6 |
2.51 |
11.4 |
1.99 |
5.1 |
0.14 |
Forskolin / 1 |
107.8 |
1.52 |
109.3 |
1.17 |
105.0 |
1.55 |
Forskolin / 10 |
107.0 |
2.43 |
108.5 |
4.17 |
102.4 |
2.58 |
Prochloraz / 0.1 |
98.3 |
2.20 |
101.2 |
0.47 |
100.7 |
0.86 |
Prochloraz / 1 |
99.4 |
1.19 |
101.5 |
0.78 |
99.5 |
2.37 |
Test Material Plate |
||||||
TM / 0.0001 |
92.0 |
1.49 |
95.4 |
1.94 |
96.0 |
3.19 |
TM / 0.001 |
93.7 |
2.77 |
97.7 |
1.98 |
97.9 |
0.70 |
TM / 0.01 |
96.4 |
2.19 |
99.8 |
2.92 |
96.9 |
1.73 |
TM / 0.1 |
97.1 |
1.52 |
100.8 |
1.27 |
93.7 |
0.69 |
TM / 1 |
49.8* |
1.35 |
94.8 |
4.36 |
40.6* |
4.18 |
TM / 10 |
1.2* |
0.54 |
6.6* |
0.89 |
1.1* |
0.48 |
TM / 100 |
2.4* |
2.28 |
7.5* |
3.35 |
2.9* |
2.31 |
TM =Test material
*Indicates cytotoxicity greater than 20 %. These concentrations were omitted from further analysis
Table 2: QC Plate Raw Data and Fold Change Results - Testosterone
|
Average Testosterone (pg/mL) |
Testosterone Fold Change over SC |
Testosterone Fold Change over SC-Background Subtracted |
||||||
Concentration |
Run 2 |
Run 3 |
Run 4 |
Run 2 |
Run 3 |
Run 4 |
Run 2 |
Run 3 |
Run 4 |
Background |
1135 |
1073 |
1262 |
0.45 |
0.57 |
0.54 |
N/A |
N/A |
N/A |
Blank |
2581 |
1873 |
2304 |
1.03 |
1.00 |
0.99 |
1.05 |
1.00 |
0.97 |
DMSO |
2511 |
1874 |
2339 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1 μM Forskolin |
3764 |
2595 |
3120 |
1.50 |
1.38 |
1.33 |
1.91 |
1.90 |
1.73 |
10 μM Forskolin |
4751 |
3019 |
3780 |
1.89 |
1.61 |
1.62 |
2.63 |
2.43 |
2.34 |
0.1 μM Prochloraz |
1816 |
1149 |
1818 |
0.72 |
0.61 |
0.78 |
N/A |
N/A |
N/A |
1 μM Prochloraz |
1188 |
501 |
1153 |
0.47 |
0.27 |
0.49 |
N/A |
N/A |
N/A |
For forskolin induction of testosterone, background hormone concentration is subtracted from all other concentrations prior to calculating fold change values.
N/A = not applicable
Table 3: QC Plate Raw Data and Fold Change Results - Estradiol
|
Average Estradiol (pg/mL) |
Estradiol Fold Change over SC |
||||
Concentration |
Run 2 |
Run 3 |
Run 4 |
Run 2 |
Run 3 |
Run 4 |
Background |
41 |
56 |
77 |
0.21 |
0.31 |
0.35 |
Blank |
208 |
182 |
228 |
1.04 |
1.01 |
1.02 |
DMSO |
200 |
179 |
223 |
1.00 |
1.00 |
1.00 |
1 μM Forskolin |
1343 |
1254 |
1538 |
6.72 |
7.01 |
6.91 |
10 μM Forskolin |
2116 |
1661 |
2477 |
10.60 |
9.28 |
11.13 |
0.1 μM Prochloraz |
133 |
104 |
171 |
0.67 |
0.58 |
0.77 |
1 μM Prochloraz |
57 |
52 |
83 |
0.29 |
0.29 |
0.37 |
Table 4: Quality Control Plate Results for Testosterone and Estradiol
|
Testosterone |
Estradiol |
||||
Run 2 |
Run 3 |
Run 4 |
Run 2 |
Run 3 |
Run 4 |
|
Basal Production-Blank Wells |
2581 pg/mL |
1873 pg/mL |
2304 pg/mL |
208 pg/mL |
182 pg/mL |
228 pg/mL |
Basal Production-Solvent Control Wells |
2511 pg/mL |
1874 pg/mL |
2339 pg/mL |
200 pg/mL |
179 pg/mL |
223 pg/mL |
Induction (10 μM Forskolin) |
3 |
2 |
2 |
10.6 |
9.3 |
11.1 |
Inhibition (1 μM Prochloraz) |
0.5 |
0.3 |
0.5 |
0.3 |
0.3 |
0.4 |
For forskolin induction of testosterone, background hormone concentration is subtracted from all other concentrations prior to calculating fold change values.
Table 5: Fold Change Over the Solvent Control Results for the Test Material
Conc. (µM) |
Testosterone |
Estradiol |
||||||||||
Run 2 |
Run 3 |
Run 4 |
Run 2 |
Run 3 |
Run 4 |
|||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
0.0001 |
1.02 |
0.04 |
1.07 |
0.01 |
0.98 |
0.03 |
0.99 |
0.03 |
1.14 |
0.02 |
1.02 |
0.02 |
0.001 |
1.02 |
0.01 |
1.08 |
0.02 |
0.92* |
0.04 |
0.97 |
0.05 |
1.11 |
0.02 |
0.95 |
0.04 |
0.01 |
1.03 |
0.02 |
1.09 |
0.02 |
0.97 |
0.00 |
1.00 |
0.04 |
1.15 |
0.04 |
0.98 |
0.01 |
0.1 |
1.00 |
0.02 |
1.04 |
0.06 |
0.94 |
0.03 |
0.95 |
0.04 |
1.07 |
0.04 |
0.95 |
0.02 |
1 |
N/A |
N/A |
0.69 |
0.02 |
N/A |
N/A |
N/A |
N/A |
0.63 |
0.03 |
N/A |
N/A |
N/A = concentration not analysed due to cytotoxicity and/or contamination
*Denotes statisticalsignificance (p≤0.05)
Table 3: Results of Run 2 Aromatase Activity Assay
Concentration (M) |
Aromatase Activity (% of VC) |
Individual Aromatase Activity (% of VC) |
|||
Mean |
SD |
Value 1 |
Value 2 |
Value 3 |
|
Positive Control |
|||||
TA |
101.86 |
0.761 |
102.40 |
101.32 |
ND |
NSB |
0.00 |
0.012 |
0.01 |
-0.01 |
ND |
10^-5 |
0.81 |
0.063 |
0.86 |
0.77 |
ND |
10^-6 |
6.62 |
0.139 |
6.72 |
6.53 |
ND |
10^-6.5 |
17.72 |
1.153 |
18.53 |
16.90 |
ND |
10^-7 |
37.17 |
0.349 |
37.41 |
36.92 |
ND |
10^-7.5 |
65.52 |
0.258 |
65.34 |
65.71 |
ND |
10^-8 |
84.07 |
0.789 |
84.63 |
83.52 |
ND |
10^-9 |
95.38 |
0.107 |
95.30 |
95.46 |
ND |
10^-10 |
96.36 |
1.895 |
97.70 |
95.02 |
ND |
Test Material |
|||||
TA |
98.14 |
0.131 |
98.05 |
98.23 |
ND |
NSB |
0.00 |
0.024 |
0.02 |
-0.02 |
ND |
10^-3 |
76.55 |
2.301 |
79.16 |
74.80 |
75.71 |
10^-3.5 |
ND |
ND |
ND |
ND |
ND |
10^-4 |
94.09 |
0.409 |
94.45 |
93.64 |
94.19 |
10^-4.5 |
ND |
ND |
ND |
ND |
ND |
10^-5 |
89.43 |
8.186 |
80.15 |
95.63 |
92.51 |
10^-6 |
96.25 |
6.628 |
99.33 |
100.77 |
88.64 |
10^-7 |
98.10 |
1.287 |
97.60 |
97.13 |
99.56 |
10^-8 |
100.12 |
1.459 |
101.67 |
99.92 |
98.77 |
10^-9 |
97.70 |
0.753 |
97.90 |
96.86 |
98.33 |
10^-10 |
96.18 |
3.876 |
98.13 |
91.72 |
98.70 |
VC = Vehicle Control
TA = Full Activity Control
NSB = Background Activity Control
SD = Standard Deviation
ND = Not Determined
Table 4: Results of Run 3 Aromatase Activity Assay
Concentration (M) |
Aromatase Activity (% of VC) |
Individual Aromatase Activity (% of VC) |
|||
Mean |
SD |
Value 1 |
Value 2 |
Value 3 |
|
Positive Control |
|||||
TA |
102.50 |
0.870 |
101.88 |
103.11 |
ND |
NSB |
0.01 |
0.050 |
-0.03 |
0.05 |
ND |
10^-5 |
0.92 |
0.011 |
0.91 |
0.92 |
ND |
10^-6 |
8.26 |
0.599 |
8.68 |
7.84 |
ND |
10^-6.5 |
21.14 |
0.482 |
21.48 |
20.79 |
ND |
10^-7 |
41.23 |
1.092 |
40.46 |
42.00 |
ND |
10^-7.5 |
69.29 |
0.022 |
69.28 |
69.31 |
ND |
10^-8 |
87.26 |
1.125 |
86.46 |
88.05 |
ND |
10^-9 |
99.44 |
0.554 |
99.05 |
99.84 |
ND |
10^-10 |
104.53 |
0.100 |
104.46 |
104.60 |
ND |
Test Material |
|||||
TA |
97.50 |
1.103 |
96.72 |
98.28 |
ND |
NSB |
-0.01 |
0.011 |
-0.02 |
0.00 |
ND |
10^-3 |
ND |
ND |
ND |
ND |
ND |
10^-3.5 |
94.00 |
1.746 |
92.18 |
94.16 |
95.66 |
10^-4 |
97.06 |
2.282 |
94.45 |
98.68 |
98.06 |
10^-4.5 |
ND |
ND |
ND |
ND |
ND |
10^-5 |
99.55 |
0.878 |
99.36 |
98.78 |
100.50 |
10^-6 |
98.96 |
0.776 |
99.86 |
98.49 |
98.53 |
10^-7 |
98.63 |
2.455 |
100.69 |
95.92 |
99.29 |
10^-8 |
98.23 |
3.562 |
102.06 |
95.02 |
97.59 |
10^-9 |
79.18 |
30.077 |
94.10 |
98.89 |
44.56 |
10^-10 |
95.31 |
2.905 |
98.65 |
93.85 |
93.42 |
VC = Vehicle Control
TA = Full Activity Control
NSB = Background Activity Control
SD = Standard Deviation
ND = Not Determined
Table 5: Results of Run 4 Aromatase Activity Assay
Concentration (M) |
Aromatase Activity (% of VC) |
Individual Aromatase Activity (% of VC) |
|||
Mean |
SD |
Value 1 |
Value 2 |
Value 3 |
|
Positive Control |
|||||
TA |
102.32 |
1.854 |
103.63 |
101.01 |
ND |
NSB |
0.02 |
0.025 |
0.00 |
0.03 |
ND |
10^-5 |
1.04 |
0.135 |
1.13 |
0.94 |
ND |
10^-6 |
7.81 |
0.264 |
8.00 |
7.63 |
ND |
10^-6.5 |
20.70 |
0.092 |
20.77 |
20.64 |
ND |
10^-7 |
42.06 |
1.197 |
42.90 |
41.21 |
ND |
10^-7.5 |
68.55 |
0.602 |
68.12 |
68.97 |
ND |
10^-8 |
87.40 |
1.228 |
86.53 |
88.27 |
ND |
10^-9 |
95.55 |
3.750 |
92.90 |
98.21 |
ND |
10^-10 |
102.09 |
0.761 |
102.62 |
101.55 |
ND |
Test Material |
|||||
TA |
97.68 |
2.879 |
99.72 |
95.64 |
ND |
NSB |
-0.02 |
0.031 |
-0.04 |
0.01 |
ND |
10^-3 |
ND |
ND |
ND |
ND |
ND |
10^-3.5 |
ND |
ND |
ND |
ND |
ND |
10^-4 |
99.49 |
3.954 |
94.98 |
101.13 |
102.35 |
10^-4.5 |
99.20 |
2.165 |
99.85 |
100.97 |
96.79 |
10^-5 |
102.10 |
2.044 |
100.05 |
102.10 |
104.14 |
10^-6 |
99.90 |
3.264 |
98.68 |
97.42 |
103.60 |
10^-7 |
100.29 |
2.542 |
97.38 |
102.09 |
101.40 |
10^-8 |
98.10 |
1.765 |
97.70 |
100.03 |
96.56 |
10^-9 |
97.27 |
0.428 |
97.33 |
97.67 |
96.82 |
10^-10 |
97.92 |
1.427 |
99.53 |
97.40 |
96.83 |
VC = Vehicle Control
TA = Full Activity Control
NSB = Background Activity Control
SD = Standard Deviation
ND = Not Determined
Table 2: Results of First Valid Binding Assay
Substance |
Concentration (Log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
-3* |
72.4* |
1.7* |
-4 |
75.4 |
25.4 |
|
-5 |
105.2 |
3.5 |
|
-6 |
104.6 |
5.8 |
|
-7 |
105.2 |
2.2 |
|
-8 |
99.8 |
2.9 |
|
-9 |
106.3 |
4.6 |
|
-10 |
106.3 |
0.8 |
|
R1881 |
-7.48 |
0.0 |
0.7 |
-8.48 |
4.9 |
0.6 |
|
-9.48 |
28.5 |
1.3 |
|
-10.48 |
84.4 |
0.7 |
|
-11.48 |
96.7 |
3.1 |
|
-12.48 |
104.7 |
2.7 |
|
Dexamethasone |
-4 |
28.3 |
2.2 |
-5 |
85.1 |
3.5 |
|
-6 |
103.0 |
1.9 |
|
-7 |
95.0 |
3.7 |
|
-8 |
105.8 |
2.7 |
|
-9 |
106.7 |
3.3 |
|
-10 |
109.2 |
1.8 |
|
-11 |
105.4 |
1.8 |
*Data not assessed due to precipitation
Table 3: Results of Second Valid Binding Assay
Substance |
Concentration (Log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
-4* |
83.0* |
2.0* |
-5 |
98.2 |
4.5 |
|
-6 |
101.4 |
2.1 |
|
-7 |
101.0 |
2.0 |
|
-8 |
99.7 |
1.8 |
|
-9 |
98.2 |
2.1 |
|
-10 |
103.0 |
1.0 |
|
-11 |
100.4 |
3.2 |
|
R1881 |
-6 |
0.0 |
1.2 |
-7 |
1.6 |
0.8 |
|
-8 |
7.8 |
1.6 |
|
-9 |
47.2 |
2.9 |
|
-10 |
85.3 |
1.8 |
|
-11 |
95.6 |
3.5 |
|
Dexamethasone |
-3 |
4.3 |
0.4 |
-4 |
28.5 |
3.6 |
|
-5 |
76.1 |
3.0 |
|
-6 |
95.5 |
3.8 |
|
-7 |
102.3 |
2.9 |
|
-8 |
98.7 |
3.3 |
|
-9 |
105.5 |
3.0 |
|
-10 |
98.9 |
6.1 |
*Data not assessed due to precipitation
Table 4: Results of Third Valid Binding Assay
Substance |
Concentration (Log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
-4* |
78.3 |
4.1 |
-5 |
88.9 |
12.5 |
|
-6 |
103.4 |
1.9 |
|
-7 |
102.2 |
2.8 |
|
-8 |
99.1 |
1.6 |
|
-9 |
102.6 |
1.6 |
|
-10 |
96.7 |
4.6 |
|
-11 |
106.8 |
0.4 |
|
R1881 |
-6 |
0.0 |
4.1 |
-7 |
0.5 |
3.3 |
|
-8 |
8.2 |
1.0 |
|
-9 |
50.3 |
1.3 |
|
-10 |
91.9 |
8.1 |
|
-11 |
102.1 |
0.4 |
|
Dexamethasone |
-3 |
9.6 |
7.3 |
-4 |
27.3 |
3.0 |
|
-5 |
77.0 |
0.5 |
|
-6 |
102.2 |
3.1 |
|
-7 |
102.2 |
4.9 |
|
-8 |
104.7 |
5.5 |
|
-9 |
104.5 |
0.6 |
|
-10 |
103.9 |
4.2 |
*Data not assessed due to precipitation
Table 5: Upper and Lower Parameters in Competitive Assay Binding Curves for the Standards
Parameter |
Unit |
Run 1 |
Run 2 |
Run 3 |
|||
R1881 |
Dexamethasone |
R1881 |
Dexamethasone |
R1881 |
Dexamethasone |
||
Bottom Plateau Level |
% Binding |
0 |
N/A |
0 |
-1 |
0 |
7 |
Top Plateau Level |
% Binding |
102 |
105 |
96 |
101 |
103 |
104 |
Hill Slope |
Log10(M)^-1 |
-1.0 |
-0.6 |
-1.0 |
-0.9 |
-1.0 |
-1.0 |
Table 2: Results of First Valid Binding Assay
Substance |
Concentration (log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
10^-3* |
54.8* |
3.8* |
10^-4 |
85.2 |
2.8 |
|
10^-5 |
94.3 |
3.3 |
|
10^-6 |
99.3 |
2.4 |
|
10^-7 |
99.7 |
2.2 |
|
10^-8 |
103.2 |
2.1 |
|
10^-9 |
99.1 |
3.3 |
|
10^-10 |
97.9 |
1.0 |
|
Estradiol (NSB) |
10^-7 |
0.0 |
0.4 |
10^-8 |
7.3 |
0.7 |
|
10^-8.5 |
22.2 |
1.8 |
|
10^-9 |
46.8 |
3.1 |
|
10^-9.5 |
74.5 |
1.3 |
|
10^-10 |
86.4 |
3.5 |
|
10^-11 |
97.3 |
1.9 |
|
19-Norethindrone |
10^-4 |
1.9 |
0.5 |
10^-4.5 |
6.3 |
0.3 |
|
10^-5.5 |
42.1 |
0.6 |
|
10^-6 |
68.8 |
2.4 |
|
10^6.5 |
96.0 |
0.6 |
|
10^-7 |
93.8 |
2.7 |
|
10^-7.5 |
95.7 |
3.9 |
|
10^-8.5 |
94.8 |
2.4 |
|
Octyltriethoxysilane |
10^-3 |
90.2 |
1.3 |
10^-4 |
92.3 |
2.4 |
|
10^-5 |
99.1 |
1.9 |
|
10^-6 |
100.3 |
2.9 |
|
10^-7 |
100.1 |
3.7 |
|
10^-8 |
102.3 |
1.0 |
|
10^-9 |
102.9 |
1.3 |
|
10^-10 |
102.0 |
6.7 |
*Data not assessed due to precipitation
Table 3: Results of Second Valid Binding Assay
Substance |
Concentration (log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
10^-3* |
66.3* |
5.1* |
10^-4 |
91.9 |
1.2 |
|
10^-5 |
98.2 |
2.9 |
|
10^-6 |
101.2 |
2.3 |
|
10^-7 |
102.3 |
2. |
|
10^-8 |
98.7 |
3.8 |
|
10^-9 |
103.8 |
1.4 |
|
10^-10 |
100.5 |
0.6 |
|
Estradiol (NSB) |
10^-7 |
0.0 |
0.1 |
10^-8 |
2.3 |
0.3 |
|
10^-8.5 |
7.5 |
0.2 |
|
10^-9 |
20.4 |
0.7 |
|
10^-9.5 |
47.2 |
3.3 |
|
10^-10 |
73.5 |
2.4 |
|
10^-11 |
98.4 |
2.5 |
|
19-Norethindrone |
10^-4 |
2.6 |
0.3 |
10^-4.5 |
8.3 |
0.0 |
|
10^-5.5 |
48.1 |
1.3 |
|
10^-6 |
73.9 |
1.8 |
|
10^6.5 |
89.8 |
2.5 |
|
10^-7 |
99.7 |
1.9 |
|
10^-7.5 |
100.2 |
2.0 |
|
10^-8.5 |
100.0 |
6.7 |
|
Octyltriethoxysilane |
10^-3 |
80.4 |
6.3 |
10^-4 |
97.2 |
2.7 |
|
10^-5 |
102.3 |
3.0 |
|
10^-6 |
100.2 |
1.1 |
|
10^-7 |
102.8 |
2.6 |
|
10^-8 |
100.3 |
2.5 |
|
10^-9 |
102.3 |
4.3 |
|
10^-10 |
99.7 |
3.4 |
*Data not assessed due to precipitation
Table 4: Results of Third Valid Binding Assay
Substance |
Concentration (log[M]) |
Specific Binding (%) |
Standard Deviation |
Test Material |
10^-4 |
90.4 |
12.1 |
10^-5 |
104.6 |
2.5 |
|
10^-6 |
97.4 |
2.1 |
|
10^-7 |
94.4 |
9.5 |
|
10^-8 |
95.8 |
3.1 |
|
10^-9 |
95.6 |
3.0 |
|
10^-10 |
88.8 |
10.1 |
|
10^-11 |
95.1 |
6.2 |
|
Estradiol (NSB) |
10^-7 |
0.0 |
0.4 |
10^-8 |
7.5 |
1.1 |
|
10^-8.5 |
22.5 |
0.5 |
|
10^-9 |
44.8 |
4.7 |
|
10^-9.5 |
73.6 |
7.4 |
|
10^-10 |
92.5 |
10.8 |
|
10^-11 |
97.4 |
3.4 |
|
19-Norethindrone |
10^-4 |
1.4 |
0.5 |
10^-4.5 |
7.0 |
0.4 |
|
10^-5.5 |
45.7 |
0.4 |
|
10^-6 |
70.4 |
5.7 |
|
10^6.5 |
88.3 |
2.4 |
|
10^-7 |
93.4 |
2.8 |
|
10^-7.5 |
98.1 |
1.5 |
|
10^-8.5 |
101.1 |
3.0 |
|
Octyltriethoxysilane |
10^-3 |
92.5 |
4.9 |
10^-4 |
103.7 |
3.5 |
|
10^-5 |
111.8 |
12.0 |
|
10^-6 |
101.4 |
2.2 |
|
10^-7 |
102.0 |
2.5 |
|
10^-8 |
100.8 |
2.3 |
|
10^-9 |
97.7 |
2.5 |
|
10^-10 |
98.0 |
2.2 |
Table 5: Upper and Lower Parameters in Competitive Assay Binding Curves for the Standards
Parameter |
Unit |
Run 1 |
Run 2 |
Run 3 |
|||
17β-estradiol |
19-norethindrone |
17β-estradiol |
19-norethindrone |
17β-estradiol |
19-norethindrone |
||
Log e(Syx) |
- |
0.78 |
1.28 |
0.55 |
1.01 |
1.64 |
0.95 |
Bottom Plateau Level |
% Binding |
-1 |
2 |
0 |
0 |
0 |
-3 |
Top Plateau Level |
% Binding |
98 |
97 |
102 |
101 |
98 |
100 |
Hill Slope |
Log10(M)^-1 |
-1.0 |
-1.3 |
-1.0 |
-1.0 |
-1.0 |
-0.9 |
Table 2: Preliminary Cytotoxicity Assay
Test Material Concentration (M) |
Run 1 |
Run 2 |
Run 3 |
|||
Cell Viability (% of VC) |
Cell Viability (% of VC) |
Cell Viability (% of VC) |
||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
10^-10 |
96 |
2 |
98 |
5 |
105 |
6 |
10^-9 |
90 |
9 |
96 |
6 |
105 |
4 |
10^-8 |
87 |
8 |
94 |
5 |
106 |
4 |
10^-7 |
87 |
3 |
94 |
6 |
104 |
3 |
10^-6 |
84 |
3 |
92 |
7 |
98 |
5 |
10^-5 |
60* |
3* |
66* |
2* |
71* |
3* |
10^-4 |
10* |
3* |
17* |
3* |
24* |
2* |
10^-3 |
40* |
3* |
41* |
3* |
48* |
2* |
VC = Vehicle control
*Viability <80 % and considered to be cytotoxic
Table 3: Results of the First Valid Transcriptional Activation Assay
Substance |
Concentration (M) |
RTA (% of PC) |
RTA with ICI |
Cell Viability (% of VC) |
|||
Mean |
SD |
Value 1 |
Value2 |
Mean |
SD |
||
Test Material |
10^-12 |
-0.5 |
1.5 |
0.1 |
-0.1 |
94.0 |
6.1 |
10^-11 |
-2.1 |
1.1 |
0.2 |
-0.1 |
94.4 |
8.5 |
|
10^-10 |
-1.3 |
2.0 |
0.0 |
0.0 |
87.4 |
5.2 |
|
10^-9 |
-0.9 |
1.7 |
0.1 |
-0.1 |
87.4 |
5.9 |
|
10^-8 |
3.6 |
3.1 |
0.2 |
0.1 |
90.1 |
5.6 |
|
10^-7 |
2.0 |
2.5 |
0.2 |
0.1 |
91.1 |
7.8 |
|
10^-6 |
1.1 |
0.8 |
0.1 |
0.0 |
83.0 |
6.2 |
|
10^-5 |
* |
* |
0.1* |
0.1* |
50.9* |
3.7* |
|
17β-Estradiol |
10^-15 |
-0.1 |
1.2 |
0.1 |
-0.1 |
97.9 |
14.5 |
10^-14 |
-1.5 |
1.8 |
0.1 |
0.0 |
89.3 |
11.7 |
|
10^-13 |
* |
* |
0.1* |
0.0* |
80.0* |
6.4* |
|
10^-12 |
10.2 |
2.7 |
0.1 |
0.0 |
85.4 |
12.1 |
|
10^-11 |
84.2 |
20.8 |
0.4 |
0.2 |
88.5 |
10.4 |
|
10^-10 |
102.5 |
10.4 |
0.3 |
0.1 |
84.1 |
11.3 |
|
10^-9 |
* |
* |
0.3* |
0.2* |
77.7* |
8.6* |
|
10^-8 |
* |
* |
0.6* |
0.2* |
72.3* |
6.8* |
|
17α-Estradiol |
10^-13 |
0.8 |
1.7 |
0.1 |
0.0 |
102.4 |
5.8 |
10^-12 |
0.0 |
2.2 |
0.2 |
0.0 |
94.6 |
2.9 |
|
10^-11 |
9.4 |
3.8 |
0.1 |
0.0 |
88.7 |
4.9 |
|
10^-10 |
46.3 |
9.1 |
0.1 |
-0.1 |
94.2 |
1.3 |
|
10^-9 |
100.6 |
15.3 |
0.4 |
0.2 |
93.8 |
6.6 |
|
10^-8 |
90.8 |
10.7 |
0.4 |
0.1 |
90.5 |
6.3 |
|
10^-7 |
97.5 |
17.1 |
0.9 |
0.4 |
87.9 |
3.6 |
|
10^-6 |
* |
* |
19.9* |
3.6* |
74.4* |
6.9* |
|
Corticosterone |
10^-11 |
-0.1 |
1.4 |
0.0 |
0.0 |
100.8 |
14.1 |
10^-10 |
-1.6 |
1.1 |
0.1 |
-0.1 |
94.5 |
10.1 |
|
10^-9 |
-1.1 |
1.7 |
0.1 |
-0.1 |
95.2 |
12.3 |
|
10^-8 |
-0.5 |
1.8 |
0.1 |
-0.1 |
94.4 |
6.3 |
|
10^-7 |
1.1 |
1.6 |
0.3 |
0.0 |
101.4 |
13.0 |
|
10^-6 |
1.0 |
1.2 |
0.2 |
0.0 |
94.1 |
7.2 |
|
10^-5 |
3.1 |
1.5 |
0.1 |
0.0 |
97.9 |
10.3 |
|
10^-4 |
* |
* |
0.0* |
-0.1* |
70.9* |
5.6* |
|
17α-Methyltestosterone |
10^-12 |
0.6 |
1.2 |
0.1 |
-0.1 |
105.5 |
8.6 |
10^-11 |
1.9 |
0.9 |
0.1 |
-0.1 |
107.2 |
6.4 |
|
10^-10 |
28.6 |
17.9 |
0.1 |
-0.1 |
105.8 |
6.2 |
|
10^-9 |
83.0 |
24.1 |
0.1 |
-0.1 |
106.2 |
7.7 |
|
10^-8 |
105.0 |
28.6 |
0.4 |
0.1 |
109.6 |
9.9 |
|
10^-7 |
83.0 |
12.6 |
0.4 |
0.1 |
100.3 |
9.7 |
|
10^-6 |
92.6 |
17.6 |
0.3 |
0.1 |
100.7 |
6.6 |
|
10^-5 |
79.6 |
25.8 |
0.1 |
0.0 |
101.3 |
2.5 |
|
RTA = Relative Transcriptional Activation
PC = Positive Control (1 nM 17β-Estradiol)
VC = Vehicle Control
SD = Standard Deviation
*Cytotoxicity observed, data not evaluated
Table 4: Results of the Second Valid Transcriptional Activation Assay
Substance |
Concentration (M) |
RTA (% of PC) |
RTA with ICI |
Cell Viability (% of VC) |
|||
Mean |
SD |
Value 1 |
Value2 |
Mean |
SD |
||
Test Material |
10^-8.5 |
-4.6 |
4.2 |
0.0 |
0.0 |
98.7 |
8.3 |
10^-8 |
-7.4 |
3.5 |
0.0 |
0.0 |
98.5 |
9.4 |
|
10^-7.5 |
-7.4 |
3.5 |
0.0 |
0.0 |
98.2 |
7.3 |
|
10^-7 |
-5.3 |
2.5 |
0.0 |
-0.1 |
94.3 |
9.5 |
|
10^-6.5 |
-1.7 |
3.8 |
0.2 |
0.0 |
94.4 |
8.9 |
|
10^-6 |
-6.8 |
2.6 |
0.1 |
0.1 |
87.1 |
4.9 |
|
10^-5.5 |
* |
* |
0.3* |
0.4* |
68.1* |
5.5* |
|
10^-5 |
* |
* |
0.5* |
0.4* |
54.7* |
3.9* |
|
17β-Estradiol |
10^-15 |
-5.6 |
2.8 |
-0.1 |
0.0 |
95.6 |
12.2 |
10^-14 |
-8.2 |
4.4 |
-0.1 |
0.1 |
94.5 |
19.7 |
|
10^-13 |
-2.7 |
5.3 |
0.0 |
0.0 |
100.1 |
2.8 |
|
10^-12 |
30.9 |
7.3 |
-0.1 |
-0.1 |
89.7 |
15.5 |
|
10^-11 |
104.1 |
17.6 |
0.0 |
0.1 |
88.8 |
16.6 |
|
10^-10 |
119.9 |
20.5 |
0.0 |
0.0 |
98.4 |
3.5 |
|
10^-9 |
149.7 |
16.5 |
-0.1 |
0.0 |
95.8 |
3.4 |
|
10^-8 |
116.7 |
21.0 |
0.4 |
0.5 |
90.9 |
4.5 |
|
17α-Estradiol |
10^-13 |
-2.6 |
2.2 |
0.0 |
0.0 |
97.1 |
5.7 |
10^-12 |
-7.5 |
3.5 |
0.0 |
0.0 |
98.6 |
7.2 |
|
10^-11 |
-2.0 |
2.7 |
0.0 |
0.0 |
96.4 |
4.4 |
|
10^-10 |
17.5 |
1.6 |
0.0 |
0.0 |
94.4 |
9.4 |
|
10^-9 |
70.1 |
9.7 |
0.1 |
0.1 |
93.1 |
4.0 |
|
10^-8 |
108.3 |
8.0 |
0.2 |
0.1 |
91.7 |
4.5 |
|
10^-7 |
130.0 |
17.9 |
0.1 |
0.1 |
91.0 |
6.6 |
|
10^-6 |
107.6 |
38.9 |
1.6 |
1.9 |
82.7 |
4.1 |
|
Corticosterone |
10^-11 |
-4.4 |
2.7 |
0.0 |
0.0 |
98.8 |
4.6 |
10^-10 |
-8.2 |
2.1 |
0.0 |
0.0 |
100.0 |
6.9 |
|
10^-9 |
-5.5 |
4.0 |
0.1 |
0.0 |
95.9 |
6.0 |
|
10^-8 |
-7.2 |
2.1 |
0.0 |
0.0 |
93.8 |
6.2 |
|
10^-7 |
-5.5 |
2.1 |
0.2 |
0.0 |
93.0 |
4.5 |
|
10^-6 |
-6.1 |
2.0 |
0.1 |
0.0 |
97.2 |
5.1 |
|
10^-5 |
-1.8 |
2.1 |
0.1 |
0.0 |
92.9 |
6.2 |
|
10^-4 |
* |
* |
0.0* |
0.0* |
76.7* |
5.7 |
|
17α-Methyltestosterone |
10^-12 |
-3.6 |
2.1 |
0.0 |
0.0 |
103.6 |
5.9 |
10^-11 |
-7.2 |
3.1 |
0.0 |
0.0 |
103.8 |
6.4 |
|
10^-10 |
2.1 |
4.0 |
0.0 |
-0.1 |
98.4 |
7.4 |
|
10^-9 |
33.2 |
3.6 |
0.0 |
-0.1 |
97.0 |
7.4 |
|
10^-8 |
104.0 |
10.0 |
0.1 |
0.0 |
94.9 |
5.8 |
|
10^-7 |
103.3 |
13.1 |
0.1 |
0.0 |
93.4 |
8.4 |
|
10^-6 |
124.3 |
9.4 |
0.0 |
-0.1 |
94.7 |
5.7 |
|
10^-5 |
107.8 |
5.7 |
0.0 |
0.0 |
82.6 |
3.9 |
|
RTA = Relative Transcriptional Activation
PC = Positive Control (1 nM 17β-Estradiol)
VC = Vehicle Control
SD = Standard Deviation
*Cytotoxicity observed, data not evaluated
Table 1: Summary of fertilisation success and fecundity during the 21-day exposure of fathead minnow
Time-Weighted Average Concentration (μg a.i./L) |
Replicate |
Fertilisation Success (% Viable) |
Fecundity (Number Eggs/Female/Day) |
Control |
A |
96 |
15 |
B |
96 |
3 |
|
C |
93 |
9 |
|
D |
97 |
13 |
|
Mean |
96 (1.8) |
10 (5.5) |
|
Solvent Control |
A |
95 |
10 |
B |
92 |
7 |
|
C |
96 |
16 |
|
D |
98 |
19 |
|
Mean |
95 (2.8) |
13 (5.1) |
|
Pooled Control |
|
95 (2.2) |
12 (5.2) |
0.20 |
A |
98 |
8 |
B |
81 |
6 |
|
C |
98 |
12 |
|
D |
93 |
11 |
|
Mean |
92 (7.8) |
9 (2.5) |
|
1.7 |
A |
95 |
15 |
B |
82 |
14 |
|
C |
98 |
14 |
|
D |
96 |
12 |
|
Mean |
93 (7.7) |
14 (1.3) |
|
18 |
A |
96 |
10 |
B |
97 |
8 |
|
C |
94 |
3 |
|
D |
95 |
10 |
|
Mean |
96 (1.4) |
8 (3.6) |
Standard deviation presented in parentheses
Table 2: Male termination endpoint summary during the 21-day exposure
Time-Weighted Average Concentration (μg a.i./L) |
Replicate |
Mean Tubercle Score |
Mean Body Weight (g) |
Mean GSI (%) |
Control |
A |
39 |
3.6918 |
1.11 |
B |
18 |
3.5832 |
1.85 |
|
C |
32 |
2.8077 |
1.64 |
|
D |
26 |
2.4467 |
1.79 |
|
Mean |
29 (8.9) |
3.1324 (0.6033) |
1.60 (0.30) |
|
Solvent Control |
A |
28 |
3.2258 |
2.21 |
B |
38 |
3.4667 |
1.23 |
|
C |
38 |
4.1788 |
1.38 |
|
D |
44 |
3.4229 |
1.73 |
|
Mean |
37 (6.5) |
3.5735 (0.4169) |
1.64 (0.44) |
|
Pooled Control |
|
33 (8.4) |
3.3529 (0.5348) |
1.62 (0.36) |
0.20 |
A |
31 |
3.5521 |
1.49 |
B |
34 |
3.1199 |
1.19 |
|
C |
33 |
3.3679 |
1.09 |
|
D |
39 |
2.8006 |
1.51 |
|
Mean |
34 (3.4) |
3.2101 (0.3254) |
1.32 (0.21) |
|
1.7 |
A |
39 |
2.7166 |
1.31 |
B |
31 |
3.6322 |
1.46 |
|
C |
32 |
3.2209 |
2.31 |
|
D |
26 |
3.5945 |
1.19 |
|
Mean |
32 (5.5) |
3.2910 (0.4256) |
1.57 (0.51) |
|
18 |
A |
33 |
2.8446 |
1.30 |
B |
34 |
3.3862 |
1.89 |
|
C |
12 |
3.2532 |
1.76 |
|
D |
33 |
2.5302 |
1.11 |
|
Mean |
28 (11) |
3.0036 (0.3908) |
1.52 (0.37) |
Standard deviation presented in parentheses.
Table 3: Female termination endpoint summary during the 21-day exposure
Time-Weighted Average Concentration (μg a.i./L) |
Replicate |
Mean Body Weight (g) |
Mean GSI (%) |
Control |
A |
1.4355 |
12.87 |
B |
1.3599 |
15.80 |
|
C |
1.2649 |
14.86 |
|
D |
1.6055 |
11.74 |
|
Mean |
1.4164 (0.1441) |
13.82 (1.85) |
|
Solvent Control |
A |
1.7579 |
11.73 |
B |
1.4115 |
12.13 |
|
C |
1.6264 |
12.23 |
|
D |
1.3812 |
10.72 |
|
Mean |
1.5442 (0.1794) |
11.70 (0.69) |
|
Pooled Control |
|
1.4803 (0.1654) |
12.76 (1.72) |
0.20 |
A |
1.6600 |
7.11 |
B |
1.6528 |
10.89 |
|
C |
1.6678 |
10.93 |
|
D |
1.6746 |
12.45 |
|
Mean |
1.6638 (0.0095) |
10.34 (2.28) |
|
1.7 |
A |
1.5784 |
12.95 |
B |
1.4769 |
11.07 |
|
C |
1.5482 |
12.97 |
|
D |
1.6367 |
11.53 |
|
Mean |
1.5600 (0.0665) |
12.13 (0.98) |
|
18 |
A |
1.6786 |
11.64 |
B |
1.5141 |
13.88 |
|
C |
1.6197 |
14.65 |
|
D |
1.7729 |
11.13 |
|
Mean |
1.6463 (0.1084) |
12.82 (1.70) |
Table 4: Results of vitellogenin analysis during the 21-day exposure
Time-Weighted Average Concentration (μg a.i./L) |
Replicate |
Male VTG Concentration (ng/mL) |
Female VTG Concentration (ng/mL) |
Control |
A |
131 |
2 276 477 |
B |
154 |
2 447 436 |
|
C |
311 |
1 476 051 |
|
D |
359 |
1 804 223 |
|
Mean |
239 |
2 001 047 |
|
Solvent Control |
A |
122 |
2 240 962 |
B |
187 |
4 815 294 |
|
C |
37 |
2 384 604 |
|
D |
11 |
1 360 770 |
|
Mean |
89 |
2 700 407 |
|
Pooled Control |
|
164 |
2 350 727 |
0.20 |
A |
145 |
3 152 010 |
B |
60 |
3 927 760 |
|
C |
83 |
3 038 580 |
|
D |
36 |
1202 |
|
Mean |
81 |
2 529 888 |
|
1.7 |
A |
133 |
2 815 041 |
B |
2215 |
2 788 055 |
|
C |
114 |
2 645 575 |
|
D |
3039 |
3 737 020 |
|
Mean |
1375 |
2 996 423 |
|
18 |
A |
940 |
3 398 292 |
B |
12 585 |
2 557 112 |
|
C |
24 |
4 222 792 |
|
D |
669 |
2 493 083 |
|
Mean |
3554 |
3 167 820 |
Table 1: Summary of endpoints (developmental stage, snout-vent length, hind limb length and whole body wet weight) following 7 days of exposure
Conc. (µg a.i./L) |
Rep. |
DS |
SV Length (mm) |
HL Length (mm) |
Normalised* HL Length (mm) |
Whole Body ww (g) |
||||||||
Median |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
||
Control |
A |
54 |
18.83 |
0.61 |
3.2 |
2.69 |
0.25 |
9.2 |
0.14 |
0.014 |
10 |
0.3589 |
0.0300 |
8.4 |
B |
54 |
18.68 |
0.86 |
4.6 |
2.80 |
0.31 |
11 |
0.15 |
0.015 |
9.8 |
0.3623 |
0.0312 |
8.6 |
|
C |
54 |
17.66 |
1.42 |
8.1 |
2.58 |
0.34 |
13 |
0.15 |
0.0093 |
6.4 |
0.3267 |
0.0636 |
19 |
|
D |
54 |
18.62 |
1.24 |
6.7 |
2.52 |
0.38 |
15 |
0.14 |
0.016 |
12 |
0.3838 |
0.0684 |
18 |
|
Mean |
54 |
18.45 |
1.03 |
5.6 |
2.65 |
0.32 |
12 |
0.14 |
0.014 |
9.5 |
0.3579 |
0.0483 |
14 |
|
0.088 |
A |
54 |
19.07 |
0.45 |
2.3 |
2.88 |
0.37 |
13 |
0.15 |
0.017 |
11.6 |
0.4004 |
0.0646 |
16 |
B |
54 |
19.00 |
1.47 |
7.7 |
2.65 |
0.21 |
7.8 |
0.14 |
0.0077 |
5.5 |
0.3854 |
0.0836 |
22 |
|
C |
54 |
19.91 |
0.72 |
3.6 |
2.81 |
0.17 |
5.9 |
0.14 |
0.0041 |
2.9 |
0.4482 |
0.0420 |
9.4 |
|
D |
54 |
19.35 |
1.66 |
8.6 |
2.80 |
0.31 |
11 |
0.14 |
0.011 |
7.5 |
0.4194 |
0.0781 |
19 |
|
Mean |
54 |
19.33 |
1.07 |
5.6 |
2.79 |
0.26 |
9.4 |
0.14 |
0.010 |
6.9 |
0.4134 |
0.0671 |
16 |
|
0.87 |
A |
54 |
19.23 |
1.33 |
6.9 |
2.90 |
0.31 |
11 |
0.15 |
0.007 |
4.8 |
0.4299 |
0.0826 |
19 |
B |
54 |
18.83 |
1.39 |
7.4 |
2.55 |
0.52 |
21 |
0.13 |
0.019 |
14 |
0.3792 |
0.0690 |
18 |
|
C |
54 |
19.81 |
1.85 |
9.3 |
2.80 |
0.51 |
18 |
0.14 |
0.015 |
11 |
0.4304 |
0.0937 |
22 |
|
D |
54 |
18.84 |
1.40 |
7.4 |
2.56 |
0.25 |
10 |
0.14 |
0.0071 |
5.2 |
0.4003 |
0.0767 |
19 |
|
Mean |
54 |
19.18 |
1.49 |
7.8 |
2.70 |
0.40 |
15 |
0.14 |
0.012 |
9 |
0.4009 |
0.0805 |
20 |
|
8.7 |
A |
54 |
18.28 |
1.47 |
8.0 |
2.56 |
0.22 |
8.7 |
0.14 |
0.014 |
10.1 |
0.3627 |
0.0729 |
20 |
B |
54 |
18.31 |
1.70 |
9.3 |
2.42 |
0.38 |
16 |
0.13 |
0.013 |
9.7 |
0.3613 |
0.0840 |
23 |
|
C |
54 |
17.77 |
0.88 |
4.9 |
2.53 |
0.30 |
12 |
0.14 |
0.014 |
9.7 |
0.3408 |
0.0555 |
16 |
|
D |
54 |
17.71 |
1.99 |
11 |
2.39 |
0.41 |
17 |
0.13 |
0.0087 |
6.5 |
0.3368 |
0.1081 |
32 |
|
Mean |
54 |
18.02 |
1.51 |
8.4 |
2.47 |
0.33 |
13 |
0.14 |
0.012 |
9.0 |
0.3504 |
0.0801 |
23 |
|
Conc. = Mean measured concentration
Rep. = Replicate
DS = Developmental stage
SV = Snout-Vent
HL = Hindlimb
*Normalised by snout-vent length
ww = wet weight
Table 2: Summary of endpoints (developmental stage, snout-vent length, hind limb length and whole body wet weight) following 21 days of exposure
Conc. (µg a.i./L) |
Rep. |
DS |
SV Length (mm) |
HL Length (mm) |
Normalised* HL Length (mm) |
Whole Body ww (g) |
||||||||
Median |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
Mean |
SD |
CV (%) |
||
Control |
A |
60 |
17.54 |
3.49 |
20 |
24.38 |
1.80 |
7.4 |
0.73 |
0.19 |
25 |
0.9345 |
0.1475 |
16 |
B |
60 |
14.91 |
3.41 |
23 |
26.41 |
1.19 |
4.5 |
0.57 |
0.15 |
26 |
1.1208 |
0.1208 |
11 |
|
C |
60 |
18.44 |
4.63 |
25 |
24.05 |
2.79 |
12 |
0.79 |
0.27 |
34 |
0.9709 |
0.1829 |
19 |
|
D |
61 |
18.93 |
3.79 |
20 |
23.90 |
2.53 |
11 |
0.81 |
0.22 |
27 |
0.9333 |
0.1752 |
19 |
|
Mean |
60 |
17.46 |
3.83 |
22 |
34.69 |
2.08 |
8.5 |
0.73 |
0.21 |
28 |
0.9899 |
0.1566 |
16 |
|
0.088 |
A |
60 |
16.69 |
3.87 |
23 |
25.03 |
2.06 |
8.2 |
0.67 |
0.19 |
28 |
0.9682 |
0.1612 |
17 |
B |
60 |
17.64 |
4.94 |
28 |
24.38 |
2.77 |
11 |
0.75 |
0.28 |
37 |
0.9947 |
0.2127 |
21 |
|
C |
60 |
18.59 |
4.76 |
26 |
24.04 |
2.51 |
10 |
0.80 |
0.27 |
34 |
0.9552 |
0.1616 |
17 |
|
D |
60 |
18.79 |
4.25 |
23 |
24.69 |
2.32 |
9.4 |
0.78 |
0.24 |
30 |
1.0167 |
0.1744 |
17 |
|
Mean |
60 |
17.93 |
4.46 |
25 |
24.53 |
2.41 |
10 |
0.75 |
0.24 |
32 |
0.9837 |
0.1775 |
18 |
|
0.87 |
A |
60 |
16.76 |
4.59 |
27 |
24.76 |
2.26 |
9.1 |
0.69 |
0.25 |
36 |
1.0083 |
0.1738 |
17 |
B |
61 |
19.49 |
4.80 |
25 |
24.01 |
3.02 |
13 |
0.84 |
0.29 |
34 |
1.0117 |
0.2328 |
23 |
|
C |
60 |
18.04 |
3.15 |
17 |
24.23 |
2.68 |
11 |
0.76 |
0.20 |
26 |
0.9435 |
0.2078 |
22 |
|
D |
60 |
17.98 |
4.51 |
25 |
24.54 |
2.91 |
12 |
0.73 |
0.24 |
32 |
0.9645 |
0.2368 |
25 |
|
Mean |
60 |
18.07 |
4.27 |
24 |
24.39 |
2.72 |
11 |
0.76 |
0.24 |
32 |
0.9820 |
0.2128 |
22 |
|
8.7 |
A |
60 |
15.92 |
4.57 |
29 |
25.22 |
2.24 |
8.9 |
0.65 |
0.23 |
36 |
1.0869 |
0.1711 |
16 |
B |
60 |
17.54 |
3.56 |
20 |
25.29 |
1.71 |
6.7 |
0.70 |
0.18 |
25 |
1.0638 |
0.1403 |
13 |
|
C |
60 |
16.36 |
5.48 |
33 |
25.20 |
2.21 |
8.8 |
0.66 |
0.25 |
38 |
1.0358 |
0.2063 |
20 |
|
D |
60 |
16.41 |
4.28 |
26 |
24.80 |
2.32 |
9.4 |
0.68 |
0.23 |
34 |
1.0208 |
0.1491 |
15 |
|
Mean |
60 |
16.56 |
4.47 |
27 |
25.13 |
2.12 |
8.4 |
0.67 |
0.22 |
33 |
1.0518 |
0.1667 |
16 |
|
Conc. = Mean measured concentration
Rep. = Replicate
DS = Developmental stage
SV = Snout-Vent
HL = Hindlimb
*Normalised by snout-vent length
ww = wet weight
Description of key information
A series of studies were conducted in line with those recommended in Tier 1 of the US EPA Endocrine Screening strategy and are discussed in the attached weight of evidence (WoE) document. These did not indicate any endocrine activity that would impact on the human health risk assessment for propargite.
The following studies are included in this section of the dataset:
- Hershberger Assay (Rat), OCSPP 890.1400, OECD 441, Davis 2011
- Pubertal Female Assay (Rat), OCSPP 890.1450 and Pubertal Male Assay (Rat), OCSPP 890.1500, Davis 2011
- Uterotrophic Assay (Rat) OCSPP 890.1600, OECD 440, Davis 2011
- Steroidogenesis Assay (Human cell line H295R), OCSPP 890.1550, OECD 456, Wagner 2011
- Aromatase Assay (human recombinant microsomes), OCSPP 890.1200, Wilga 2011
- AR Binding Assay (Rat prostate cytosol), OCSPP 890.1150, Willoughby 2011
- ER Binding Assay (Rat uterine cytosol), OCSPP 890.1250, Willoughby 2011
- ERα Transcriptional Activation Assay (Human cell line HeLa 9903), OCSPP 890.1300, OECD 455, Willoughby 2011
- Fish Short-term Reproduction Assay (FSTRA), OCSPP 890.1350, OECD 229, York 2011
- Amphibian Metamorphosis Assay (AMA; Frog), OCSPP 890.1100, OECD 231, Lee 2011
Additional information
The Endocrine Disruptor Screening Program’s (EDSP) Tier 1 assay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signalling pathways. This interaction includes agonism and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in the weight of evidence (WoE) assessment prepared by the EPA and attached to this summary.
In determining whether propargite interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was present.
On 3 December 2013, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a Weight-of Evidence (WoE) analysis of the potential interaction of propargite with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in the attached report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).
For propargite, for the estrogen pathway, the Tier 1 in vitro assays were negative. In the female pubertal assay, the only estrogen-related effect observed in the absence of overt toxicity was an increase in the incidence and severity of interstitial cell hyperplasia of the ovaries (7/16 rats versus 0/16 in controls) at the low dose (62.5 mg/kg/day). There were no estrogen-related effects observed in the mammalian Part 158 toxicity studies. In the fish short-term reproduction assay (FSTRA), while fecundity was lower than guideline criteria and ovarian histopathological alterations were observed in both the controls and treatments which lessen the degree of confidence in this study, there were limited effects observed in this study. A delay in ovary maturation stage was observed at the high concentration and a decrease in gonadal somatic index (GSI) at the low treatment. Overall, there is a lack of convincing evidence for potential interaction with the estrogen pathway.
For the androgen pathway, the Tier 1 in vitro assays were negative. In the male pubertal assay, the only effect observed in the absence of overt toxicity was a decrease in serum testosterone levels seen at the low dose (62.5 mg/kg/day). In the Hershberger assay, there was a dose-dependent decrease in androgen responsive tissue weights; ventral prostate and LABC weights were decreased at all doses as well as effects on seminal vesicles at the high dose (150 mg/kg/day); however the effects at the high dose were in the presence of overt toxicity (>10 % decrease in body weight). No androgen-related effects were observed in the Part 158 studies. In the FSTRA, again, while gonadal histopathological alterations were observed in both controls and treatment groups, there were no effects observed in the male fish. Therefore, given the lack of responses in the in vitro assays, the male pubertal study and the FSTRA, there is no convincing evidence of potential interaction with the androgen pathway.
Thyroid-related effects were observed in the male and female pubertal assays in the absence of overt toxicity, as well as in the amphibian metamorphosis assay (AMA). Increased follicular cell height and decreased colloidal area were observed in the male and female pubertal assays, while increased follicular cell hyperplasia and hypertrophy were observed in the AMA. Serum T4 levels were also increased in the male and female pubertal assays. While the T4 increase in the females was not statistically significant, it was of similar magnitude to the T4 increase in males.
In the EDSP Tier 1 assays, there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in the young animals.
For propargite, the point of departure (POD) of 4 mg/kg/day used for the current human health risk assessment is based on systemic toxicity characterised as increased mortality, decreases in body weight/body weight gain and food consumption seen at 19 mg/kg/day (LOAEL) in a chronic toxicity/carcinogenicity study. This POD is used for deriving the chronic reference dose (RfD) for chronic dietary risk assessment and calculating the Margins of Exposure (MOEs) for non-dietary risk assessments.
In general, since the POD and the toxicity endpoints of concern are based on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young. However in the case of propargite although no data exists for potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended since: 1) the POD is approximately 15-fold lower than the dose (62.5 mg/kg/day) that caused thyroid lesions in the EDSP Tier 1 pubertal assays; 2) no thyroid-related effects were seen in any of the mammalian toxicity studies; 3) the CTA would be tested at higher doses would not be expected to yield a POD lower than the level currently used for risk assessment; 4) the RfD and the MOEs are protective of the thyroid effect seen in the pubertal assays. Therefore, mammalian EDSP Tier 2 testing is NOT recommended for propargite since additional testing is not expected to impact EPA’s current regulatory point of departures and endpoints for human health risk assessments.
In the AMA, dose-responsive effects on thyroid gland histopathology reported in the absence of overt toxicity suggest that propargite may have the potential to interact with the HPT axis in amphibians (i.e., increase in the incidence of mild follicular cell hyperplasia and a marked increase in the incidence of mild follicular cell hypertrophy and mild colloid depletion and changes in colloid quality in the highest concentration). Additionally, somewhat similar histological effects as well as changes in thyroid hormones were noted in the mammalian pubertal studies. The current chronic toxicity endpoint for fish (surrogates for aquatic-phase amphibians) is 16 μg/L (NOAEC) based on a reductions in survival in a fish early-life cycle.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
