Chlorpyrifos

Immunotoxicity

Description of key information

Study Type	Species	Findings	Guideline	Reliability
28-Day Rat Diet	Rat	NOAEL: 10 mg/kg/day; no humoral immune response	EPA OPPTS 870.7800	1

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

immunotoxicity: sub-chronic oral

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:

EPA OPPTS 870.7800

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419, TSN101285
Purity: 99.8%

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

female

Route of administration:

oral: feed

Vehicle:

acetone

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuous

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Dose / conc.:

2 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

A cage-side examination was conducted at least once a day, at approximately the same time each day (usually in the morning). Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study.

All rats were weighed pre-exposure, twice weekly throughout the study, and at necropsy (terminal).

Feed consumed was determined pre-exposure, twice during the first week and at least weekly thereafter for all animals by weighing feed containers at the start and end of a measurement cycle.

The following clinical chemistry parameters were evaluated: Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLT) count, Reticulocyte (RET) count. RBC indices measured included: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC).

Red Blood Cell (RBC) and brain cholinesterase activity were measured.

Sacrifice and pathology:

A limited gross necropsy was conducted on all animals (including positive control animals) by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The eyes were examined in situ by application of a moistened microscope slide to each cornea. All animals had the head removed, although the vehicle control and treated animals also had the cranial cavity opened and the brain removed, rinsed with saline, and blotted dry. The brain was then dissected into the right and left hemispheres. The right hemisphere was weighed. Both sections were individually quick frozen in liquid nitrogen (see the assessment of brain acetyl cholinesterase activity section of protocol for further details). The skin, on all animals, was thenvcreflected from the carcass, the thoracic and abdominal cavities opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. For all study animals (including the positive control animals) spleen and thymus were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated.

Specific cell-mediated immunity:

The immunotoxic potential of chlorpyrifos was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBC) using an enzyme-linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti-SRBC IgM.

Positive control:

Cyclophosphamide monohydrate

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related observations in the ranked parameters throughout the study. Clinical observations consisted of a few animals in the
low and high dose group with dermatitis. These observations were considered spurious and unrelated to treatment due to the isolated occurrence and lack of dose response.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Details on results:

There were no treatment related effects on body weight, body weight gain, feed consumption, hematology or absolute and relative spleen and thymus weights. RBC and brain cholinesterase activities were inhibited by chlorpyrifos in a dose-dependent manner. The response for RBCs was inhibited to a greater extent and at lower dose levels when compared to the response for the brain. RBC cholinesterase activity was significantly decreased at all dose levels when compared to the vehicle control and values were 53.7%, 0.9% and 0.2% of the control value for the 0.4, 2, and 10 mg/kg/day dose groups, respectively. Brain cholinesterase activity was unchanged in the 0.4 mg/kg/day dose group, relative to control, but was significantly decreased in the intermediate and high-dose groups. Brain cholinesterase activities were 91.3% and 18.2% of the control value for the 2 and 10 mg/kg/day dose groups, respectively. Analysis of the anti-SRBC IgM ELISA data did not reveal a statistically significant difference between the three doses of chlorpyrifos and the vehicle control. Despite the lack of statistical significance, a 64% and 41% decrease in the mean anti-SRBC IgM concentration, relative to control, was observed for the 2 and 10 mg/kg/day dose groups, respectively. The interpretation of the biological significance of these decreases was confounded by several factors including the biological variability in the control group, the lack of a clear dose response, and the lack of any correlative evidence on other immune parameters (e.g., spleen and thymus weights and hematology). Based on these considerations, a clear assessment of the antibody response could not be made, and it was determined that the evidence for the immunotoxicity potential of chlorpyrifos was equivocal.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no immunotox effects at highest dose tested

Conclusions:

A clear assessment of the antibody response could not be made, and it was determined that the evidence for the immunotoxicity potential of chlorpyrifos was equivocal.

Executive summary:

Ten female Crl:CD(SD) rats per group were given test diets formulated to supply 0, 0.4, 2.0, or 10 mg/kg/day chlorpyrifos for 28 days to evaluate the potential for immunotoxicity. Actual time-weighted average doses were 0, 0.416, 2.13, or 10.7 mg/kg/day, respectively. An additional group of ten female Crl:CD(SD) rats exposed to 0 mg/kg/day was included to serve as positive immunosuppressive controls and received intraperitoneal doses of 20 mg/kg cyclophosphamide (CP) on study days 24-28. The immunotoxic potential of chlorpyrifos was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBC) using an enzyme-linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti-SRBC IgM. Additional parameters evaluated included daily cage-side observations, weekly detailed clinical observations, body weights, feed consumption, hematology, selected organ weights, gross examinations of selected tissues and red blood cell (RBC) and brain cholinesterase activities.

 

There were no treatment related effects on body weight, body weight gain, feed consumption, hematology or absolute and relative spleen and thymus weights. RBC and brain cholinesterase activities were inhibited by chlorpyrifos in a dose-dependent manner. The response for RBCs was inhibited to a greater extent and at lower dose levels when compared to the response for the brain. RBC cholinesterase activity was significantly decreased at all dose levels when compared to the vehicle control and values were 53.7%, 0.9% and 0.2% of the control value for the 0.4, 2, and 10 mg/kg/day dose groups, respectively. Brain cholinesterase activity was unchanged in the 0.4 mg/kg/day dose group, relative to control, but was significantly decreased in the intermediate and high-dose groups. Brain cholinesterase activities were 91.3% and 18.2% of the control value for the 2 and 10 mg/kg/day dose groups, respectively.

Analysis of the anti-SRBC IgM ELISA data did not reveal a statistically significant difference between the three doses of chlorpyrifos and the vehicle control. Despite the lack of statistical significance, a 64% and 41% decrease in the mean anti-SRBC IgM concentration, relative to control, was observed for the 2 and 10 mg/kg/day dose groups, respectively. The interpretation of the biological significance of these decreases was confounded by several factors including the biological variability in the control group, the lack of a clear dose response, and the lack of any correlative evidence on other immune parameters (e.g., spleen and thymus weights and hematology). Based on these considerations, a clear assessment of the antibody response could not be made, and it was determined that the evidence for the immunotoxicity potential of chlorpyrifos was equivocal.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Effect on immunotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a rat immunotoxicity study, diets formulated to supply 0, 0.4, 2.0, or 10 mg/kg/day chlorpyrifos for 28 days was provided. There were no treatment related effects on body weight, body weight gain, feed consumption, hematology or absolute and relative spleen and thymus weights. RBC and brain cholinesterase activities were inhibited by chlorpyrifos in a dose-dependent manner. The response for RBCs was inhibited to a greater extent and at lower dose levels when compared to the response for the brain. RBC cholinesterase activity was significantly decreased at all dose levels when compared to the vehicle control and values were 53.7%, 0.9% and 0.2% of the control value for the 0.4, 2, and 10 mg/kg/day dose groups, respectively. Brain cholinesterase activity was unchanged in the 0.4 mg/kg/day dose group, relative to control, but was significantly decreased in the intermediate and high-dose groups. Brain cholinesterase activities were 91.3% and 18.2% of the control value for the 2 and 10 mg/kg/day dose groups, respectively. Based on these considerations, a clear assessment of the antibody response could not be made, and it was determined that the evidence for the immunotoxicity potential of chlorpyrifos was equivocal.

Justification for classification or non-classification

The test substance did not produce a humoral response in rats in a 28-day immunotoxicity study. Therefore, the substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.