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Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1400 (2009)
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Remarks:
Hershberger bioassay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
10 days
Frequency of treatment:
once daily
Dose / conc.:
0.1 mg/kg bw/day
Remarks:
antiandrogenic study
Dose / conc.:
0.25 mg/kg bw/day
Remarks:
androgenic and antiandrogenic studies
Dose / conc.:
0.5 mg/kg bw/day
Remarks:
androgenic and antiandrogenic studies
Dose / conc.:
1 mg/kg bw/day
Remarks:
androgenic study
No. of animals per sex per dose:
6 (in both studies)
Control animals:
yes, concurrent vehicle
Positive control:
Testosterone Propionate (androgenic study); Flutamide (antiandrogenic study)
Details on results:
The test substance did not affect androgen sensitive organ weights consistent with potential androgenic or antiandrogenic activity in the Hershberger Bioassay when administered to castrated male rats up to 1.0 mg/kg/day for 10 days.
Conclusions:
The test substance did not affect androgen sensitive organ weights consistent with potential androgenic or antiandrogenic activity in the Hershberger Bioassay when administered to castrated male rats up to 1.0 mg/kg/day for 10 days.
Executive summary:

The objective of this study was to evaluate the potential androgenic and antiandrogenic effects of the test substance when administered by oral gavage to castrated rats for 10 days. The study was conducted according to the guideline, U. S. EPA OPPTS 890.1400.

The study was separated into 2 separate studies, one designed to detect androgenic (i.e., androgen receptor agonist-like activity) and one to detect antiandrogenic (i.e., androgen receptor antagonist-like activity). In the androgenic study, four groups of young adult castrated Crl:CD(SD) rats (6/group) were dosed by oral gavage with 0, 0.25, 0.5, or 1.0 mg/kg/day of the test substance for 10 consecutive days and sacrificed approximately 24 hours after the last administered dose. A separate castrated positive control group, administered 0.4 mg/kg/day of the androgen receptor agonist testosterone propionate (TP), was included to verify test system performance. In the antiandrogenic study, four groups of young adult castrated Crl:CD(SD) rats (6/group) were dosed by oral gavage with 0, 0.1, 0.25, or 0.5 mg/kg/day of the test substance for 10 consecutive days and sacrificed approximately 24 hours after the last administered dose. A separate castrated positive control group, administered 3 mg/kg/day of the androgen receptor antagonist flutamide (FT), was included to verify test system performance. In addition to the test substance or positive control treatment, all treatment groups also received a daily injection of 0.4 mg/kg of the reference androgen receptor agonist, TP.

Body weights and clinical observations were recorded daily; food consumption was recorded once over the duration of the study (test days 9-10). At necropsy, organ weights (liver, ventral prostate, seminal vesicle [plus fluids and coagulating glands], levator ani-bulbocavernosus muscle, paired Cowper’s glands and the glans penis) were collected. The Cowper’s gland and ventral prostate were saved for possible microscopic evaluation, and serum was saved for possible hormonal analysis. Microscopic evaluation of the Cowper’s glands was performed in the antiandrogenic study. Hormonal analyses in both the androgenic and antiandrogenic studies, and microscopic evaluation of the Cowper’s glands in the androgenic study were deemed unnecessary. The potential androgenic activity of the test substance was evaluated by its ability to increase weights for the androgen-dependent tissues, similar to the effects induced by the positive control, TP. The potential antiandrogenic activity of the test substance was evaluated by its ability to decrease the TP-stimulated weight increases for the androgen-dependent tissues, similar to the effects induced by the positive control, FT.

In both the androgenic and antiandrogenic studies, performance criteria for confirmation of a valid test, as specified in the test guideline, were met. In both the androgenic and antiandrogenic studies, no test substance-related deaths occurred, and there were no clinical observations noted during the inlife phase of the study. No test substance-related effects on body weight or nutritional parameters were observed, with the exception of a slight, non statistically significant decrease in food efficiency observed in rats administered 0.5 and 1 mg/kg/day in the androgenic study. There were no test substance-related effects indicative of androgenic or antiandrogenic activity. At necropsy, there were no test substancerelated effects on organ weights, and there were no gross observations noted. In the antiandrogenic study, microscopic evaluation of the Cowper’s glands showed no test substance-related effects.

Although there were no test substance-related effects observed in the current studies, the doses used for this study reached a maximum-tolerated dose as determined in previous studies in adult rats. In those studies, there was decreased cholinesterase activity and clinical signs of toxicity observed at 2 mg/kg, and at 0.5 mg/kg/day, there was decreased cholinesterase activity in the absence of clinical signs of toxicity. Therefore, while no test substance-related effects were observed in the current study, cholinesterase activity should be decreased at dosages of 0.5 and 1 mg/kg/day.

For the androgenic study in rats administered 0.4 mg/kg/day of the positive control chemical, TP, no deaths occurred, and there were no clinical observations noted during the in-life phase of the study. Mean body weight gain was statistically significantly increased over the duration of the study (test days 0 to 10), and this increase in weight gain was accompanied by an increase in mean daily food consumption. However, there were no statistically significant effects on mean final body weight observed on any test day, and there were no effects on food efficiency. As expected, rats administered TP showed effects consistent with an androgen receptor agonist. There were statistically significant increases in the weights for all the androgen-dependent tissues evaluated (ventral prostate, seminal vesicle [plus fluids and coagulating glands], levator ani-bulbocavernosus muscle, paired Cowper’s glands, and the glans penis). There were no gross observations noted at necropsy.

For the antiandrogenic study in rats administered 3 mg/kg/day of the positive control chemical, FT administered concurrently with 0.4 mg/kg/day of the reference chemical TP, no deaths occurred, and there were no clinical observations noted during the in-life phase of the study. No effects on body weight or nutritional parameters were observed. As expected, rats administered FT showed effects consistent with an androgen receptor antagonist. There were statistically significant decreases in the TP-stimulated weights for all the androgen-dependent tissues evaluated (ventral prostate, seminal vesicle [plus fluids and coagulating glands], levator ani-bulbocavernosus muscle, paired Cowper’s glands, and the glans penis). There were no gross observations noted at necropsy. Microscopic evaluation of the Cowper’s glands showed mild atrophy in 5 rats and moderate atrophy in one rat, consistent with the expected effect from the positive control.

In conclusion, the test substance did not show any effects on any parameters that were consistent with potential androgenic or antiandrogenic effects in castrated male rats. Therefore, under the conditions of this study, the test substance did not induce androgenic or antiandrogenic activity in the Hershberger Bioassay when administered up to 1.0 mg/kg/day for 10 consecutive days.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 890.1450
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Remarks:
female pubertal assay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Postnatal day (PND) 22 to 42
Frequency of treatment:
once daily
Dose / conc.:
0.5 mg/kg bw/day
Dose / conc.:
1 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on results:
The test substance did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function when administered orally to peripubertal female rats at dosage levels of 0.5 and 1.0 mg/kg/day
Conclusions:
The test substance did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function when administered orally to peripubertal female rats at dosage levels of 0.5 and 1.0 mg/kg/day
Executive summary:

The objective of this study was to assess the potential effects of the test substance on the endocrine system by identifying effects on pubertal development and thyroid function in the juvenile/peripubertal female rat. The protocol was designed to be in compliance with the OPPTS Guideline 890.1450.

The test substance in the vehicle (deionized water) was administered by oral gavage to 2 groups of 15 peripubertal female Crl:CD(SD) rats, obtained from non-treated time-mated females, once daily during

postnatal day (PND) 22 to 42. Dosage levels were 0.5 and 1.0 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 15 peripubertal females received only the

vehicle on a comparable regimen.

All females selected for study were observed twice daily for mortality and moribundity from weaning through study termination. Clinical observations and body weights were recorded daily. All females were observed daily for vaginal opening. Once vaginal opening was observed, daily vaginal lavages were performed for each female to determine the stage of the estrous cycle. A complete necropsy was conducted on all rats on PND 42; selected organs were weighed and preserved. Hormone (thyroxine [T4] and thyroid stimulating hormone [TSH]) and clinical pathology evaluations (urea nitrogen and creatinine) were conducted on all animals. In addition, histopathological evaluation of the thyroid, kidney, ovary, and uterus was performed.

All animals survived to the scheduled necropsy on PND 42. There were no clinical or macroscopic findings observed at any dosage level.

No test substance-related effects were noted on mean body weights or body weight gains in the 0.5 and 1.0 mg/kg/day groups. Mean ages and body weights at the age of attainment of vaginal opening, the mean age at the first occurrence of estrus, estrous cyclicity, and mean estrous cycle length in these groups were unaffected by test substance administration. Although the mean age at attainment of vaginal opening in the control group fell within the range of the performance criteria, the CV in this group (10.54) was higher than the maximum acceptable CV in the performance criteria (6.52).

No test substance-related effects on serum creatinine, urea nitrogen, T4, or TSH levels were noted at either dosage level.

There were no test substance-related effects noted on organ weights in the 0.5 and 1.0 mg/kg/day groups. No test substance-related microscopic findings were noted in these groups. The mean adrenal gland weight in the control group was lower than the acceptable range in the performance criteria; however, the CV for this value was within the acceptable range.

Based on the lack of effects on the mean age and body weight at attainment of vaginal patency, estrous cyclicity, wet and blotted uterus weights, ovary weights, and histopathology of the female reproductive organ weights, or test substance-related macroscopic and microscopic findings, the test substance did not alter the hypothalamic-pituitary-gonadal axis function when administered orally to peripubertal female rats at dose levels of 0.5 and 1.0 mg/kg/day. Based on the absence of effects on serum hormone levels, thyroid weights, and microscopic alterations in the thyroid, the test substance did not display the potential to disrupt the hypothalamic-pituitary-thyroid axis.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 890.1500
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other: Endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Postnatal day (PND) 23 to 53
Frequency of treatment:
once daily
Dose / conc.:
0.5 mg/kg bw/day
Dose / conc.:
1 mg/kg bw/day
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on results:
The test substance did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function when administered orally to peripubertal male rats at dosage levels of 0.5 and 1.0 mg/kg/day
Conclusions:
The test substance did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function when administered orally to peripubertal male rats at dosage levels of 0.5 and 1.0 mg/kg/day
Executive summary:

The objective of this study was to assess the potential effects of the test substance on the endocrine system, by identifying effects on pubertal development and thyroid function in the juvenile/peripubertal male rat. The protocol was designed to be in compliance with the OPPTS Guideline 890.1500.

The test substance in the vehicle (deionized water) was administered by oral gavage to 2 groups of 15 juvenile/peripubertal male Crl:CD(SD) rats, obtained from non-treated time-mated females, once daily during postnatal day (PND) 23 to 53. Dosage levels were 0.5 and 1.0 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 15 juvenile/peripubertal males received only the vehicle on a comparable regimen.

All males selected for study were observed twice daily for mortality and moribundity from weaning through study termination. Clinical observations and body weights were recorded daily. All males were observed daily (beginning on PND 30) for balanopreputial separation. A complete necropsy was conducted on all rats found dead or that survived to the scheduled euthanasia on PND 53; selected organs were weighed and preserved. Hormone (thyroxine [T4], thyroid stimulating hormone [TSH], and testosterone) and clinical pathology evaluations (creatinine and urea nitrogen) were conducted on all surviving animals on PND 53. In addition, histopathological evaluation of the thyroid, kidney, testis, and epididymis was performed.

One male each in the control and 1.0 mg/kg/day groups was found dead on study day 42 or 45. A perforation in the esophagus and yellow contents in the thoracic cavity were noted for the male in the 1.0 mg/kg/day group; therefore, this death was determined to be the result of an intubation error. Although the cause of death could not be determined for the male in the control group, macroscopic findings of dark red discoloration of the lungs were noted at necropsy. All other males survived to the scheduled euthanasia on PND 53.

There were no test substance-related clinical findings noted at any dosage level at the detailed physical examination or approximately 45 minutes following dose administration.

No statistically significant or test substance-related effects were noted on mean body weights or body weight gains in the 0.5 and 1.0 mg/kg/day groups. Mean ages and body weights at the age of attainment of balanopreputial separation in the 0.5 and 1.0 mg/kg/day groups were unaffected by test substance administration.

No test substance-related effects on serum creatinine, urea nitrogen, T4, testosterone, or TSH levels were noted at either dosage level.

There were no test substance-related effects noted on organ weights in the 0.5 and 1.0 mg/kg/day groups. No test substance-related macroscopic findings or microscopic changes were noted in the kidney, testes, epididymides, or thyroid gland at either dosage level.

Based on the lack of effects on the mean age and body weight at attainment of balanopreputial separation, male reproductive organs, serum testosterone levels, or test substance-related macroscopic and microscopic findings, the test substance did not alter the hypothalamic-pituitary-gonadal axis function when administered orally to peripubertal male rats at dosage levels of 0.5 and 1.0 mg/kg/day. Based on the absence of effects on serum thyroid hormone levels, thyroid weights, and microscopic alterations in the thyroid, the test substance did not display the potential to disrupt hypothalamic-pituitarythyroid axis.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1600
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo
Remarks:
uterotrophic bioassay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
3 days
Frequency of treatment:
once daily
Dose / conc.:
0.25 mg/kg bw/day
Dose / conc.:
0.5 mg/kg bw/day
Dose / conc.:
1 mg/kg bw/day
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control:
17α-Ethynyl estradiol
Details on results:
The test substance did not induce estrogenic effects in uterotrophic assay when administered up to 1 mg/kg/day of the test substance for 3 consecutive days.
Conclusions:
The test substance did not induce estrogenic effects in uterotrophic assay when administered up to 1 mg/kg/day of the test substance for 3 consecutive days.
Executive summary:

The objective of this study was to evaluate the potential estrogenic effects of the test substance when administered by oral gavage to ovariectomized rats for 3 days. The study was conducted following U.S. EPA guideline OPPTS 890.1600.

Four groups of young adult ovariectomized Crl:CD(SD) rats (6/group) were dosed by oral gavage with 0, 0.25, 0.5, or 1.0 mg/kg/day of the test substance for 3 consecutive days and sacrificed approximately 24 hours after the last administered dose. A separate ovariectomized positive control group, administered 0.1 mg/kg/day of the estrogen receptor agonist 17α-ethynyl estradiol, was included to verify test system performance. Body weights, food consumption, and clinical observations were recorded daily. Vaginal cytology was evaluated daily to assess the potential of the test substance to induce cytological changes consistent with those observed with the positive control. At necropsy, uterine weights were collected in order to assess the ability of the test substance to induce uterine growth.

No test substance-related deaths occurred, and there were no clinical observations noted during the in-life phase of the study. No test substance-related effects on body weight or nutritional parameters were observed. There were no test substance-related effects indicative of estrogenic activity. All animals receiving the test substance remained in diestrus for the duration of the study, and at necropsy, there were no gross observations noted or effects on uterine weight.

Although there were no test substance-related effects observed in the current studies, the doses used for this study reached a maximum-tolerated dose as determined in previous studies in adult rats. In those studies, there was decreased cholinesterase activity and clinical signs of toxicity observed at 2 mg/kg, and at 0.5 mg/kg/day, there was decreased cholinesterase activity in the absence of clinical signs of toxicity. Therefore, while no test substance-related effects were observed in the current study, cholinesterase activity should be decreased at dosages of 0.5 and 1 mg/kg/day.

In rats administered the positive control chemical, 17α-ethynyl estradiol, no deaths occurred, and there were no clinical observations noted during the in-life phase of the study. Mean body weight gain and mean final body weights were decreased. The body weight effects were accompanied by decreased food consumption and food efficiency. As expected, rats administered 17α-ethynyl estradiol showed effects consistent with an estrogen receptor agonist. All rats administered 17α-ethynyl estradiol showed effects on the stage of estrous, and on test day 3, all rats administered 17α-ethynyl estradiol showed cytological markers indicative of estrus. At necropsy, there were no gross observations noted. However, rats administered 17α-ethynyl estradiol showed increased uterine weights. Absolute uterine wet weight and blotted weight were increased to 313% and 266% of the negative control, respectively. Relative (to final body weight) uterine wet weight and blotted weight were increased to 344% and 297% of control, respectively. The results with 17α-ethynyl estradiol are consistent with an estrogen receptor agonist.

In conclusion, the test substance did not induce changes on any parameters consistent with the potential to act as an estrogen agonist in ovariectomized adult female rats. Under the conditions of this study, test substance did not induce estrogenic effects in the uterotrophic assay when administered up to 1 mg/kg/day for 3 consecutive days.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1200
Deviations:
no
GLP compliance:
yes
Type of method:
in vitro
Remarks:
in vitro aromatase inhibition assay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
other: human recombinant microsomes
Analytical verification of doses or concentrations:
yes
Remarks:
1E-3, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10 M
Positive control:
4-hydroxyandrostenedione (4-OH ASDN)
Details on results:
The test substance did not inhibit aromatase activity when tested at up to a maximum concentration of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in this aromatase inhibition assay.
Conclusions:
The test substance is classified as a non-inhibitor in this aromatase inhibition assay.
Executive summary:

The objective of this study was to evaluate the ability of the test substance to inhibit human recombinant microsomal aromatase activity, an enzyme responsible for the conversion of androgens to estrogens. The study was conduted according to U. S. EPA OPPTS 890.1200.

The assay is based upon the ³H2O-aromatase assay, an in vitro method that has been used extensively for the determination of the presence of the aromatase enzyme in multiple target tissues in vertebrates, and has long been used in pharmaceutical research to identify chemicals that can inhibit the catalytic activity of aromatase through an interaction with the substrate binding site on the enzyme. In brief, radioactive substrate (³H-androstenedione) and β-nicotinamide adenine dinucleotide phosphate reduced form, tetrasodium salt (NADPH), are added to microsomes containing the aromatase (CYP19) and reductase complex. ³H2O is released during the conversion of androstenedione (ASDN) to estrone, and can be quantified as a direct measurement of aromatase activity per unit reaction time. Competitive inhibition of aromatase activity by test chemicals can be detected by serial reaction tubes containing increasing concentrations of the chemical of interest.

The positive control, 4-hydroxyandrostenedione (4-OH ASDN), was evaluated to verify test system performance. As expected, 4-OH ASDN showed effects consistent with aromatase inhibition in 3 independent assays. The estimated logIC50 for 4-OH ASDN was approximately -7.2 logM.

Under the conditions of the study, the test substance did not inhibit aromatase activity when tested at up to a maximum concentration of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in this aromatase inhibition assay.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA OPPTS 890.1300
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guideline 455
Deviations:
no
GLP compliance:
yes
Type of method:
in vitro
Remarks:
transcriptional activation assay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
other: hERα-HeLa-9903 cell line
Vehicle:
other: Ultrapure water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
24 hours
Remarks:
1E-10.8, 1E-9.8, 1E-8.8, 1E-7.8, 1E-6.8, 1E-5.8, 1E-4.8, 1E-3.8 M for the first and second runs.
Details on results:
The test substance was not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.
Conclusions:
The test substance was not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system
Executive summary:

The objective of this study was to evaluate the ability of the test substance to act as an agonist of human estrogen receptor alpha (hERα) using the hERα-HeLa-9903 cell line. The study was performed according to U. S. EPA OPPTS 890.1300 and OECD guideline 455.

Preliminary assessments of cytotoxicity and precipitation were conducted in order to identify a suitable top concentration of test substance for use in the transcriptional activation assays.

Ultrapure water (water purified to 18.2 MΩ-cm) was selected as the vehicle for the test substance and did not have a significant effect on the assay. The final concentrations of test substance tested in the transcriptional activation assays were: 1E-10.8, 1E-9.8, 1E-8.8, 1E-7.8, 1E-6.8, 1E-5.8, 1E-4.8, and 1E-3.8 M for the first and second runs.

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., nonhERα- mediated) induction of the luciferase gene. The duration of exposure was 24 h. A complete concentration response curve for each of four reference compounds (17β-estradiol, 17α-estradiol, corticosterone and 17α-methyltestosterone) was run each time the transcriptional activation assay was performed.

The maximum concentration of test substance selected for use in the transcriptional activation assays was 1E-3.8 M as no cytotoxicity (≥20% reduction in cell viability) or precipitation was observed in a preliminary

cytotoxicity assay.

In two independent runs of the transcriptional activation assay, the test substance did not result in an increase in luciferase activity (RPCmax<10%) at any of the viable concentrations tested. The test substance was 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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1250
Deviations:
no
GLP compliance:
yes
Type of method:
in vitro
Remarks:
estrogen receptor binding assay
Endpoint addressed:
other: endocrine function
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
other: rat uterine cytosol
Vehicle:
other: deionized water
Analytical verification of doses or concentrations:
yes
Remarks:
1E-3, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10 M
Positive control:
19-norethindrone
Details on results:
The test substance did not competitively bind to the estrogen receptor when tested up to a maximum concentrations of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in the estrogen receptor binding assay.
Conclusions:
The test substance is classified as a non-inhibitor in the estrogen receptor binding assay.
Executive summary:

The objective of this study was to evaluate the ability of the test substance to bind to the estrogen receptors in rat uterine cytosol. The study was conducted according to the guideline U. S. EPA OPPTS 890.1250.

Saturation binding assays measure the affinity of a radiolabeled estrogen ligand, 17ß-estradiol ([3H-]E2), (Kd) for the estrogen receptor and the concentration of the estrogen receptors (Bmax) present in the cytosol. This is determined by measuring specific binding of increasing concentrations of radioligand under conditions of equilibrium. Three independent runs were performed using hexatritiated 17ßestradiol ([3H]-E2) as the radioligand to characterize the rat uterine cytosol. The Kd was approximately 0.081 nM [³H]-E2 and the Bmax was approximately 32.5 fmol/100 μg protein which are consistent with the acceptable range listed in the test guideline.

Competitive binding assays measure the binding of the radioligand to the receptors with increasing concentrations of a test substance. The concentration at which the test substance displaces half of the bound radioligand is the IC50 (often expressed as logIC50). Three independent runs were performed to evaluate methomyl for its ability to compete with [³H]-E2 in binding to rat uterine estrogen receptors in vitro. the test substance was evaluated at 8 concentrations ranging 1E-10 to 1E-3 M. Radioinert E2, the estrogen receptor agonist reference standard, 19-norethindrone, a weak estrogen receptor agonist used as the positive control, and octyltriethoxysilane, a non-estrogen receptor agonist used as the negative control, were used to verify test system performance. As expected, radioinert E2 and 19-norethindrone showed effects consistent with strong and weak competitive binding, respectively, and octyltriethoxysilane did not compete for binding to the estrogen receptor in all 3 runs. The logIC50 was determined to be approximately -9.16 and -4.85 for radioinert E2 and 19-norethindrone, respectively. The relative binding affinity of 19-norethindrone compared to radioinert E2 was approximately 0.0054%. No logIC50 values for 19-norethindrone were reported.

Under the conditions of the study, the test substance did not competitively bind to the estrogen receptor when tested up to a maximum concentrations of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in the estrogen receptor binding assay.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1550
Deviations:
no
GLP compliance:
yes
Type of method:
in vitro
Remarks:
H295R steroidogenesis assay
Endpoint addressed:
other: endocrine function
Species:
other: human cell line, H295R
Vehicle:
other: deionized water
Duration of treatment / exposure:
48 hours
Remarks:
100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM
Positive control:
forskolin
Details on results:
Negative for the induction or inhibition of steroid biosynthesis
Conclusions:
The test substance was judged to be negative for the induction or inhibition of steroid biosynthesis
Executive summary:

The objective of this study was to determine the potential for the test substance to interact with the steroidogenic pathway beginning with the sequence of reactions occurring after the gonadotropin hormone receptors (follicle stimulating hormone [FSHR] and lutinizing hormone [LHR]) through the production of testosterone and estradiol/estrone using the H295R Steroidogenesis Assay. The steroidogenic assay is not intended to identify substances that affect steroidogenesis due to effects on the hypothalamus or pituitary gland.

In a steroidogenesis assay, H295R cells cultured in vitro in 24-well plates were incubated with test substance at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 μM in triplicate for 48 hours. The test

substance’s vehicle was deionized water and its final concentration was 0.05%. Testosterone and 17ß-estradiol levels were measured by high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS). Three independent runs were performed. A Quality Control (QC) plate was included with each independent run of the test chemical to demonstrate that the assay responded properly to positive control agents at two concentration levels. Positive controls included a known inducer (forskolin) and inhibitor (prochloraz) of testosterone and 17ß-estradiol production.

The test substance did not cause statistically significant or biologically relevant changes in testosterone or 17β-estradiol synthesis relative to the vehicle control in three independent experimental runs. As expected, forskolin showed effects consistent with testosterone and 17β-estradiol induction, and prochloraz showed effects consistent with testosterone and 17β-estradiol inhibition. Performance criteria for a valid assay were met.

Under the conditions of this study, the test substance was judged to be negative for the induction or inhibition of steroid biosynthesis.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Health Effects Test Guidelines OPPTS 890.1150 (2009)
Deviations:
no
GLP compliance:
yes
Type of method:
in vitro
Remarks:
in vitro androgen receptor binding assay
Endpoint addressed:
other: endocrine
Specific details on test material used for the study:
- Substance name: Methomyl Technical
- Substance ID: DPX-X1179
- Lot#: X1179-571
- Purity: 99.4%
Species:
other: Rat prostate cytosol
Route of administration:
other: in vitro assay
Analytical verification of doses or concentrations:
yes
Remarks:
1E-3, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10 M
Positive control:
Dexamethasone
Details on results:
Negative. The test substance did not competitively bind to the androgen receptor when tested up to a maximum concentration of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in the androgen receptor binding assay.
Conclusions:
Negative
Executive summary:

The objective of this study was to evaluate the ability of the test substance to bind to the androgen receptors. The in vitro androgen receptor binding assay using rat prostate cytosol employed in this study is part of the Tier 1 battery of the Endocrine Disruptor Screening Program (EDSP), a 2-tiered approach to implement the statutory testing requirements of FFDCA section 408(p) (21 U.S.C. 346a). This assay was intended to be used in conjunction with other guidelines in the OPPTS 890 series that comprise the full screening battery under the EDSP.

The study was conducted following the guideline, U.S. EPA OPPTS 890.1150. Saturation binding assays measure the affinity of a radiolabeled androgen ligand ([3H]-R1881) (Kd) for the androgen receptor and the concentration of the androgen receptors (Bmax) present in the cytosol. This is determined by measuring specific binding of increasing concentrations of radioligand under conditions of equilibrium. Three independent runs were performed using hexatritiated R1881 ([3H]- R1881) as the radioligand to characterize the rat prostate cytosol. The Kd was approximately 0.903 nM [³H]-R1881 and the Bmax was approximately 4.5 fmol/100 μg protein which are consistent with those in the Integrated Summary Report for the androgen receptor binding assay. Competitive binding assays measure the binding of the radioligand to the receptors with increasing concentrations of a test substance. The concentration at which the test substance displaces half of the bound radioligand is the IC50 (often expressed as logIC50). Three independent runs were performed to evaluate methomyl for its ability to compete with [³H]-R1881 in binding to rat prostate androgen receptors in vitro. The test substance was evaluated at 8 concentrations between 1E-10 and 1E-3 M. Radioinert R1881, the androgen receptor agonist reference standard, and dexamethasone, a weak androgen receptor agonist used as the positive control, were used to verify test system performance.

 

As expected, radioinert R1881 showed effects consistent with strong competitive binding and dexamethasone showed effects consistent with weak competitive binding to the androgen receptor in all 3 runs. The logIC50 was determined to be approximately -9.15 and -4.41 for radioinert R1881 and dexamethasone, respectively. The relative binding affinity (RBA) of dexamethasone compared to radioinert R1881 was approximately 0.00190%. These values are consistent with those in the Integrated Summary Report. Under the conditions of the study, the test substance did not competitively bind to the androgen receptor when tested up to a maximum concentration of 1E-3 M. Therefore, the test substance is classified as a non-inhibitor in the androgen receptor binding assay.

Description of key information

Study Type Species Findings  Guideline Reliability 
Female pubertal assay  Female Rats  Did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function   

EPA OPPTS 890.1450 

 1
Peripubertal male assay  Male Rats  Did not alter the hypothalamic-pituitary-gonadal axis function and hypothalamic-pituitary-thyroid axis function  EPA OPPTS 890.1500 1
Hershberger bioassay  Male rats  Did not induce androgenic or antiandrogenic activity U.S. EPA Health Effects Test Guidelines OPPTS 890.1400 (2009) 1
Uterotrophic bioassay Female rats  Did not induce estrogenic effects U.S. EPA Health Effects Test Guidelines OPPTS 890.1600 1
in vitro androgen receptor binding assay    Rat prostate cytosol    Negative U.S. EPA Health Effects Test Guidelines OPPTS 890.1150 (2009) 1
in vitro aromatase inhibition assay  Human recombinant microsomes Non-inhibitor U.S. EPA Health Effects Test Guidelines OPPTS 890.1200 1
in vitro transcriptional activation assay  hERα-HeLa-9903 cell line not an agonist of human estrogen receptor alpha (hERα)   OECD 455, U.S. EPA OPPTS 890.1300  1
in vitro estrogen receptor binding assay  Rat uterine cytosol  non-inhibitor  U.S. EPA Health Effects Test Guidelines OPPTS 890.1250 1
H295R steroidogenesis assay  Human cell line, H295R    Negative for the induction or inhibition of steroid biosynthesis U.S. EPA Health Effects Test Guidelines OPPTS 890.1550  1

Additional information

In a variety of in vitro and in vivo tests, the test substance did not exhibit endocrine activity.