Chlorpyrifos

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

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: OPPTS 890.1150

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: endocrine

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419, TSN101285
Purity: 99.8%

Details on study design:

Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in an in vitro androgen receptor (AR) binding assay to measure the AR binding affinity of the test material. The assay measured the potential of chlorpyrifos to displace a known synthetic radiolabeled ligand (3H-R1881, methyltrienelone) from rat prostate cytosol homogenate, which is interpreted as due to the binding of the test material to the AR, presumably at the ligand binding domain. The potential of the test material to bind to the AR was assessed in independent assays at concentrations ranging from 10-10 M to 10-3 M.

Details on results:

Equivocal for AR binding, but only at in vitro concentrations (10-4 and 10-3 M) significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Conclusions:

Equivocal for AR binding, but only at in vitro concentrations (10-4 and 10-3 M) significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Executive summary:

Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in an in vitro androgen receptor (AR) binding assay to measure the AR binding affinity of the test material. The assay measured the potential of chlorpyrifos to displace a known synthetic radiolabeled ligand (3H-R1881, methyltrienelone) from rat prostate cytosol homogenate, which is interpreted as due to the binding of the test material to the AR, presumably at the ligand binding domain. The potential of the test material to bind to the AR was assessed in independent assays at concentrations ranging from 10-10M to 10-3M. The highest concentration represented the assay limit concentration. The adequacy of the experimental conditions for the detection of AR binding was confirmed by meeting quality control criteria using the following reference chemicals: R1881 (strong positive control, radioinert) and dexamethasone (weak positive control). In addition, control tubes were treated with the solvent used to dissolve the test material (i.e.,ethanol) for determination of full binding capacity and calculation of relative binding activity. At concentrations of chlorpyrifos ranging from 10-10M to 10-5M, there were no appreciable alterations in R1881 AR binding activity. At the two highest concentrations of chlorpyrifos tested, including the assay limit concentration (1 mM, 10-3 M), there was a decrease in bound radioligand that was classified as an equivocal response as described in the guideline. The results of thein vitroAR binding assay using rat prostate cytosol indicate that, under the conditions of this study, chlorpyrifos was equivocal for AR binding, but only atin vitroconcentrations (10-4and 10-3M) significantly higher thanin vivoblood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Reference 2

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: OPPTS 890.1250

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: endocrine

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419, TSN101285
Purity: 99.8%

Details on study design:

Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in an in vitro estrogen receptor (ER) binding assay to measure the estrogen receptor binding affinity of the test material. The assay measured the potential of chlorpyrifos to displace the bound reference estrogen, radiolabeled 3H-17β-estradiol, from rat uterine cytosol homogenate, which is interpreted as due to the binding of the test material to the ER, presumably at the ligand binding domain.

Details on results:

Negative

Conclusions:

Negative

Executive summary:

Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in anin vitroestrogen receptor (ER) binding assay to measure the estrogen receptor binding affinity of the test material. The assay measured the potential of chlorpyrifos to displace the bound reference estrogen, radiolabeled 3H-17β-estradiol, from rat uterine cytosol homogenate, which is interpreted as due to the binding of the test material to the ER, presumably at the ligand binding domain. The potential of the test material to bind to the ER was assessed in three independent assays at concentrations ranging from 10-10 M to 10-3 M. The highest concentration represented the assay limit concentration. The adequacy of the experimental conditions for the detection of ER binding was confirmed by meeting quality control criteria using the following reference chemicals: 17β-estradiol (strong positive control, radioinert), 19-norethindrone (weak positive control), and octyltriethoxysilane (negative control). In addition, control tubes were treated with the solvent used to dissolve the test material (i.e.,ethanol) for determination of full binding capacity and calculation of relative binding activity. At concentrations of chlorpyrifos ranging from 10-10 M to 10-3 M, there were no appreciable alterations in 17β-estradiol ER binding activity. The results of the in vitro estrogen receptor binding assay using rat uterine cytosol indicate that, under the conditions of this study, chlorpyrifos was negative (not interactive) for estrogen receptor binding at concentrations up to 10-3 M.

Reference 3

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: OPPTS 890.1300

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: endocrine

Specific details on test material used for the study:

Chlorpyrifos Technical (milled)
Lot# KC28161419, TSN101285
Purity: 99.8%

Species:

other: hERα-HeLa-9903 cell line

Details on study design:

Chlorpyrifos, (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid), was evaluated in the in vitro estrogen receptor transcriptional activation assay using the stably transfected human cell line hERα-HeLa-9903. The cell line, which has been stably transfected with human ERα and an ERα-linked luciferase reporter, detects estrogenic activity and is designed to detect hERα-mediated agonism by measuring reporter gene activation as indicated by luciferase chemiluminescence.

Details on results:

weak effect increasing estrogen receptor-mediated transactivation, but only at in vitro concentrations significantly higher than in vivo levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Conclusions:

chlorpyrifos had a weak effect increasing estrogen receptor-mediated transactivation, but only at in vitro concentrations significantly higher than in vivo levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Executive summary:

Chlorpyrifos, (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid), was evaluated in thein vitroestrogen receptor transcriptional activation assay using the stably transfected human cell line hERα-HeLa-9903. The cell line, which has been stably transfected with human ERα and an ERα-linked luciferase reporter, detects estrogenic activity and is designed to detect hERα-mediated agonism by measuring reporter gene activation as indicated by luciferase chemiluminescence. The potential of the test material to act as an ER agonist was assessed in three independent assays at concentrations ranging from 10-10 M to 10-4 M. The highest concentration was based on the limit of solubility and acceptable cytotoxicity in this assay system. The adequacy of the experimental conditions for the detection of ER agonism was confirmed with quality control criteria by employing the following reference chemicals: 17β-estradiol (strong agonist), 17α-estradiol (weak agonist), 17α-methyltestosterone (very weak agonist), and corticosterone (negative). In addition, control cultures were treated with the solvent used to dissolve the test material (i.e.,dimethyl sulfoxide) for determination of basal transcriptional activity. At the two highest concentrations of chlorpyrifos (10-5 M and 10-4 M), there was a reproducible slight increase in estrogen receptor-mediated transcriptional activity, as determined by the guideline-criterion of at least 10% of the response of the positive control (1 nM 17β-estradiol). The maximum response (RPCmax) induced by chlorpyrifos in the ER transactivation assay ranged from 10.2% to 25.4% of the response induced by the positive control. The results of thein vitroestrogen receptor transcriptional activation assay using the stably transfected human hERα-HeLa-9903 cell line indicate that, under the conditions of this study, chlorpyrifos had a weak effect increasing estrogen receptor-mediated transactivation, but only atin vitroconcentrations significantly higher thanin vivoblood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Reference 4

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: OPPTS 890.1200

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: endocrine

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419, TSN101285
Purity: 99.8%

Details on study design:

Chlorpyrifos (O,O- Diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in the in vitro human recombinant aromatase assay. The potential of the test material to inhibit aromatase activity was assessed in three independent runs at concentrations ranging from 10-10 to 10-3 M. The highest concentration was based on the 1 mM limit of the assay system.

Details on results:

non-inhibitor of aromatase activity

Conclusions:

Test material was classified as a non-inhibitor of aromatase activity.

Executive summary:

Chlorpyrifos (O,O- Diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid) was evaluated in thein vitrohuman recombinant aromatase assay. The potential of the test material to inhibit aromatase activity was assessed in three independent runs at concentrations ranging from 10-10 to 10-3 M. The highest concentration was based on the 1 mM limit of the assay system. The adequacy of the experimental conditions for detecting aromatase inhibition was confirmed by employing background and full activity controls as well as a known inhibitor of aromatase, 4-hydroxyandrostenedione, as a positive control chemical. In each run, eight concentrations of 4- hydroxyandrostenedione (in duplicate) were included as well as four background and four full activity controls. The results of the human recombinant aromatase assay with chlorpyrifos indicate that under the conditions of this study the test material was classified as a non-inhibitor of aromatase activity.

Reference 5

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: OPPTS 890.1550

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: endocrine

Specific details on test material used for the study:

Chlorpyrifos Technical (milled)
Lot# KC28161419, TSN101285
Purity: 99.8%

Details on study design:

Chlorpyrifos, (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid), was evaluated in the in vitro steroidogenesis assay using the human adrenocortical carcinoma cell line H295R. The potential of the test material to alter steroidogenesis, i.e., induce or inhibit the production of testosterone and/or estradiol was assessed in at least three independent assays at concentrations ranging from 10-10 M to 10-4 M.

Details on results:

altered steroidogenesis, but only at in vitro concentrations significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Conclusions:

Chlorpyrifos altered steroidogenesis, but only at in vitro concentrations significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Executive summary:

Chlorpyrifos, (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)ester phosphorothioic acid), was evaluated in thein vitrosteroidogenesis assay using the human adrenocortical carcinoma cell line H295R. The potential of the test material to alter steroidogenesis,i.e., induce or inhibit the production of testosterone and/or estradiol was assessed in at least three independent assays at concentrations ranging from 10-10 M to 10-4 M. The highest concentration represents the assay limit concentration of 10-4 M. The adequacy of the experimental conditions for the detection of altered steroidogenesis was confirmed with quality control criteria by employing positive control chemicals, forskolin (10-5 M) for hormone induction and prochloraz (10-6 M) for inhibition of hormone production. In addition, control cultures were treated with the solvent used to dissolve the test material (i.e.dimethyl sulfoxide) for determination of acceptable basal hormone production (i.e., minimally ~500 pg/ml testosterone and ~20 pg/ml estradiol). At concentrations of chlorpyrifos ranging from 10-10 M to 10-4 M, hormone production was reproducibly and

statistically altered at the two highest concentrations tested. Specifically, testosterone production was decreased and estradiol production was increased compared to solvent controls. The results of thein vitrosteroidogenesis assay using the human adrenocortical carcinoma cell line H295R with chlorpyrifos indicate that, under the conditions of this study, chlorpyrifos altered steroidogenesis, but only atin vitroconcentrations significantly higher thanin vivoblood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Hershberger bioassay	Male rats	Did not induce androgenic or antiandrogenic activity	U.S. EPA Health Effects Test Guidelines OPPTS 890.1400	1
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
Uterotrophic bioassay	Female rats	Did not induce estrogenic effects	U.S. EPA Health Effects Test Guidelines OPPTS 890.1600	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
in vitro estrogen receptor binding assay	Rat uterine cytosol	non-inhibitor	U.S. EPA Health Effects Test Guidelines OPPTS 890.1250	1
In Vitro Estrogen Receptor Transcriptional Activation Assay	Rat uterine cytosol	Weak effect increasing estrogen receptor-mediated transactivation, but only at in vitro concentrations significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats	U.S. EPA Health Effects Test Guidelines OPPTS 890.1300	1
in vitro androgen receptor binding assay	Rat prostate cytosol	Equivocal for AR binding, but only at in vitro concentrations (10-4 and 10-3 M) significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.	U.S. EPA Health Effects Test Guidelines OPPTS 890.1150	1
in vitro aromatase inhibition assay	Human recombinant microsomes	Non-inhibitor	U.S. EPA Health Effects Test Guidelines OPPTS 890.1200	1
H295R steroidogenesis assay	Human cell line, H295R	Altered steroidogenesis, but only at in vitro concentrations significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.	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. One study showed that Chlorpyrifos show a weak effect and another study showed equivocal effects.

 

Chlorpyrifos was evaluated in the in vitro estrogen receptor transcriptional activation assay using the stably transfected human cell line hERα-HeLa-9903. The potential of the test material to act as an ER agonist was assessed in three independent assays at concentrations ranging from 10^-10M to 10^-4M. The results of the test indicate that, under the conditions of this study, chlorpyrifos had a weak effect increasing estrogen receptor-mediated transactivation, but only at in vitro concentrations significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.

 

Chlorpyrifos was evaluated in an in vitro androgen receptor (AR) binding assay to measure the AR binding affinity of the test material. The potential of the test material to bind to the AR was assessed in independent assays at concentrations ranging from 10^-10M to 10^-3M. The results of the in vitro AR binding assay using rat prostate cytosol indicate that, under the conditions of this study, chlorpyrifos was equivocal for AR binding, but only at in vitro concentrations (10^-4and 10^-3M) significantly higher than in vivo blood levels that markedly inhibit brain and red blood cell cholinesterase activity in adult female rats.