Chlorpyrifos

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorpyrifos

EC Number:

220-864-4

EC Name:

Chlorpyrifos

Cas Number:

2921-88-2

Molecular formula:

C9H11Cl3NO3PS

IUPAC Name:

O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate

Test material form:

solid

Specific details on test material used for the study:

Lot Number: MM820905-610
Purity: 95.7%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, New York
- Age at study initiation: Approximately 40 days
- Weight at study initiation: Male: 138.6-140.7 g; Female: 112.7-115.9 g
- Housing: Housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days

DETAILS OF FOOD AND WATER QUALITY: Routine periodic analyses of the water have revealed no contaminants at levels likely to interfere with the interpretation of the results of this study. The feed was certified by Ralston Purina Co. to contain specified minimum levels of protein, fats, and fiber and to contain no more than specified maximum concentrations of certain heavy metals, chlorinated hydrocarbons, PCBs, aflatoxin, organophosphates and to have been manufactured in a plant in which the use of antibiotics and synthetic estrogens are prohibited. The levels of these contaminants in the rodent diet were judged not to be an interfering factor in the interpretation of the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 5
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Because dietary exposure of the test substance to humans and domestic animals could occur as the result of agricultural usage, administration via the diet was the route of choice for evaluation of systemic toxicity.

Vehicle:

acetone

Remarks:

and diet

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow #5002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of test substance in Purina Certified Rodent Chow #5002 was assayed on a diet mixed on study day 1. Results of the homogeneity analysis revealed homogeneous dispersion of the test material throughout the test diet.
Concentration analyses were performed on feed mixed on study day 1, study day 36, and study day 85. The results of these analyses indicate that appropriate concentrations of test substance were maintained such as to approximate the targeted dose levels closely.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Five dose levels, 0, 0.1, 1.0, 5.0, and 15 mg test substance/kg bw/day were selected. The high dose level of 15 mg/kg bw/day was expected to result in a decrease in body weight gain, produce clinical signs of ChE depression, and inhibit RBC, plasma, and brain cholinesterase activity. The intermediate dose levels of 5.0 and 1.0 mg/kg bw/day were selected to demonstrate a dose response relationship for ChE inhibition. The low dose of 0.1 mg/kg bw/day was selected to establish a no-observed-effect level since previous studies have reported no significant depression of ChE activity in rats following dietary administration of this dose for up to 2 years.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least once daily including weekends and holidays

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily including weekends and holidays

BODY WEIGHT:
- Time schedule for examinations: All animals were weighed prior to the first exposure and weekly thereafter. Cumulative body weight gains were subsequently calculated based on study day -1 weights.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumption was measured weekly, using feed jar weights, and expressed as grams/rat/day.

HAEMATOLOGY:
- Time schedule for collection of blood: Study day 90 for male animals and study day 91 for female animals
- Anaesthetic used for blood collection: Yes (light methoxyflurane anesthesia)
- Animals fasted: No
- How many animals: All rats

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At the time of scheduled necropsy
- Animals fasted: Yes
- How many animals: All surviving animals

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Study days 8, 30, 87
- Dose groups that were examined: All rats were observed in a random order and in such a manner that the observer did not know the identity of the rat or the exposure level.
- Battery of functions tested: Any unusual responses with respect to body position, activity level, coordination of movement, and gait; any unusual or bizarre behavior including but not limited to head flicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards; the presence of convulsions, tremors, increased levels of lacrimation, and/or red-colored tears, increased levels of salivation, piloerection, pupillary dilatation or constriction, unusual respiration (shallow, dyspneic, gasping, and retching) and/or mouth breathing, diarrhea, excessive or diminished urination, vocalization; hindlimb grip strength; and sensory function (audition and pain perception). Most observations were recorded after placing the rat in a clear plastic observation box measuring approximately 50 x 50 x 25 cm. If movement was not judged adequate within the allotted 20 second interval, the rat was gently prodded with the blunt end of a pencil.

OTHER: Blood for erythrocyte and plasma cholinesterase determinations was collected from nonfasted rats under light methoxyflurane anesthesia on study days 43, 90 for male animals and study days 44, 91 for female animals. At the scheduled necropsy, one-half of each animal's brain was utilized for determination of brain ChE activity, while the other half was preserved in neutral, phosphate-buffered 10% formalin.

Sacrifice and pathology:

GROSS PATHOLOGY: A gross necropsy was performed on all rats surviving the 13 weeks on test. The surviving animals were fasted overnight, weighed, anesthetized with methoxyflurane, and their tracheas clamped prior to decapitation. The rats were examined for gross pathological alterations by a veterinary pathologist.

HISTOPATHOLOGY: A histopathologic examination of the tissues was performed by a veterinary pathologist. All histopathologic examinations were performed on tissues processed in the standard manner, sectioned at 5-6 µm, and stained with hematoxylin and eosin.

Statistics:

In-life body weights and weight gains, hematologic (excluding differential WBC) and clinical chemistry parameters, fasted terminal body weights, organ weights (absolute and relative), urinary specific gravities, and cholinesterase activities were compared for differences of statistical significance using Bartlett's test for equality of variances, followed by analysis of variance (ANOVA), for parametric data, or nonparametric data. If differences were found among the groups, Dunnett's t-test or Wilcoxon's signed rank-sum test with Bonferroni's correction was used to test for differences between each treatment group and control group. Using a sequential method described by Grubbs (1969), outlying data were identified. The incidence of gross and microscopic lesions was not compared for differences of statistical significance.
Descriptive statistics (means, and standard deviations) were reported for feed consumption and white blood cell differential counts.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Several observations were noted in some of the rats during the recording of in-life observations at various times during the study. On study day 42, male rats (control and 0.1 mg/kg bw/day) were noted to have lost considerable body weight. These rats appeared healthy and no further clinical observations were recorded. Two female rats (15 mg/kg bw/day and 5 mg/kg bw/day) had damage to the right eye as the result of orbital eye bleeding procedures. All of the above observations were considered incidental and did not interfere with the conduct of the study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats administered 15 mg/kg bw/day had a statistically significant lower body weight and body weight gain relative to control at most recorded intervals. Body weight and body weight gain of female rats fed 0.1, 1, 5, or 15 mg/kg bw/day and male rats fed 0.1, 1, or 5 mg/kg bw/day were comparable to those recorded for control rats.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption among male rats fed 15 mg/kg bw/day tended to be higher than that of controls throughout the exposure period. Male rats, at lower dose levels, and female rats at all dose levels had feed consumption comparable to that recorded for control rats.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

RBC and Platelet parameters were shown to be statistically significantly different for male rats in the 5 and 15 mg/kg bw/day dose level groups. Female rats fed 15 mg/kg bw/day had statistically significant lower RBC and PCV values compared to controls. The lower RBC values were judged to most likely represent secondary effects to test substance administration rather than specific organ toxicity.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower ALP and ALT activity values were recorded for male rats fed 1, 5, and 15 mg/kg bw/day. None of these values are suggestive of damage to an internal organ, most specifically the liver, but rather to a decrease in the amount of enzyme leakage into the serum. Total protein and globulin concentrations were statistically lower in male rats fed 5 or 15 mg/kg bw/day. The albumin concentration was also lower in male rats fed 15 mg/kg bw/day. Lower serum concentrations of total protein and albumin are suggestive of a physiologic adaptation in protein metabolism in these rats.

Statistically significant lower serum glucose concentration values were identified in female rats fed 1, 5 or 15 mg/kg bw/day. These values were not judged to be toxicologically or biologically significant.

In both male and female rats fed 0.1 mg/kg bw/day, all clinical chemistry results were comparable to those of controls.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of a slight increase in urinary specific gravity values that were statistically identified in female rats given 15 mg/kg bw/day, all parameters were comparable between treated and control rats. There was no indication from histopathology or other parameters of renal damage to account for this minor change in specific gravity.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In addition to the daily recording of animal observations, functional observational batteries were performed on all rats on study days 8, 30, and 87. Male rats fed 15 mg/kg bw/day had urine staining only at the 87 day observation interval (7/10). Urine staining was noted in female rats fed 15 mg/kg bw/day on study days 8 (1/10), 30 (3/10), and 87 (8/10). On study day 87, female rats in the 1 (2/10) and 5 (2/10) mg/kg bw/day dose level groups also demonstrated urine staining. Although the exact nature and cause of urine staining was not determined, it appeared to be causally related to test substance administration.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

All values identified as statistically significant, i.e., male relative brain weight (15 mg/kg bw/day), male relative heart weight (15 mg/kg bw/day), and female absolute kidney weight (15 mg/kg bw/day) were judged to be a reflection of the terminal body weight and were not considered toxicologically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Perineal soiling, primarily seen among male and female rats fed 15 mg/kg bw/day, was the only gross observation associated with treatment. Other observations, most notably periocular hemorrhage, phthisis bulbi, and cloudy corneas, were attributed to orbital sinus bleeding procedures.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, plasma ChE was significantly lower at the interim and terminal determinations for rats given 1, 5, or 15 mg chlorpyrifos/kg bw/day. RBC (erythrocyte) ChE activity was lower at the interim determination in male rats fed 1, 5, or 15 mg/kg bw/day and lower at the terminal determination in male rats fed 5 or 15 mg/kg bw/day. Brain ChE activity was significantly lower in male rats fed 5 or 15 mg/kg bw/day. The lower ChE activity in male rats (1, 5, and 15 mg/kg bw/day) was judged an effect of test substance administration.

In female rats, plasma ChE activity at the interim determination, was statistically significantly lower in rats fed 0.1, 1, 5, and 15 mg/kg bw/day. The plasma ChE activity, at the terminal determination, was significantly lower in rats fed 1, 5, or 15 mg/kg bw/day; however, the plasma ChE for female rats of the 0.1 mg/kg bw/day dose level group was not statistically different from controls. The differences between the plasma ChE activity of the two observation periods (never greater than 20% of controls) was not considered to be of toxicologic or biologic significance. RBC (erythrocyte) ChE activity was significantly depressed at both the interim and terminal observation periods among male rats of the 1, 5, and 15 mg/kg bw/day dose level groups. As in male rats, brain ChE was significantly lower in female rats fed 5 or 15 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The only tissue alteration attributable to test substance administration occurred in male adrenal glands. Those male rats receiving 15 mg/kg bw/day had slight to moderate vacuolation, suggestive of lipid accumulation, of the zona fasiculata. Of male rats given 5 mg/kg bw/day, 8 of 10 animals had very slight vacuolation of cells in the zona fasiculata. This vacuolation was in excess of the scattered vacuolated cells normally observed in the adrenal glands of male control rats. Degenerative and inflammatory changes in the liver, kidneys, and myocardium of both male and female rats were interpreted not to be related to compound administration due principally to (1) the lack of a dose response and (2) the absence of alterations in related parameters which would be expected in a biologically or toxicologically significant event.

Effect levels

Dose descriptor:

NOEL

Effect level:

0.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

neuropathology

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (nominal)

System:

nervous system

Organ:

adrenal glands

blood

brain

Treatment related:

yes

Any other information on results incl. tables

Table-1: Cholinesterase Activity - Male

	Interim (Day 43)		Terminal (Day 90)
Dose	Plasma (U/mL)	Erythrocyte (U/mL)	Plasma (U/mL)	Erythrocyte (U/mL)	Brain (U/g)
0	0.618 ± 0.031	2.10 ± 0.42	0.640 ± 0.059	1.33 ± 0.33	11.37 ± 0.44
1	0.308 ± 0.025*	1.41 ± 0.20*	0.267 ± 0.015*	1.13 ± 0.36	11.08 ± 0.39
5	0.149 ± 0.01*	1.08 ± 0.28*	0.135 ± 0.007*	0.79 ± 0.34*	6.79 ± 0.94*
15	0.129 ± 0.006*	1.12 ± 0.36*	0.106 ± 0.013*	0.73 ± 0.29*	4.31 ± 0.27*

*Difference from control mean statistically significant; alpha = 0.05, two-sided

Table-2: Cholinesterase Activity - Female

	Interim (Day 44)		Terminal (Day 91)
Dose	Plasma (U/mL)	Erythrocyte (U/mL)	Plasma (U/mL)	Erythrocyte (U/mL)	Brain (U/g)
0	2.974 ± 0.268	1.89 ± 0.29	3.312 ± 0.209	1.81 ± 0.53	11.91 ± 0.34
1	0.539 ± 0.063*	1.19 ± 0.24*	0.619 ± 0.132*	1.16 ± 0.18*	11.77 ± 0.64
5	0.0196 ± 0.017*	0.97 ± 0.14*	0.199 ± 0.022*	0.93 ± 0.27*	7.08 ± 0.30*
15	0.154 ± 0.011	0.79 ± 0.19*	0.130 ± 0.013*	0.88 ± 0.24*	4.18 ± 0.20*

*Difference from control mean statistically significant; alpha = 0.05, two-sided

Applicant's summary and conclusion

Conclusions:

No biologically significant effects of treatment at 0.1 mg/kg bw/day in rats

Executive summary:

The test substance was fed to groups of male and female CDF Fischer-344 rats at targeted dose levels of 0 (controls), 0.1, 1, 5, or 15 mg/kg bw/day for 13 weeks. Parameters evaluated included in-life observations, functional observational batteries, body weights and body weight gains, feed consumption, hematology, urinalysis, plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity, clinical chemistries, organ weights, and gross and histopathologic alterations.

The primary effects of administration of 15 mg/kg bw/day to male rats were a depression of body weights relative to controls, depression of cholinesterase levels in the brain, plasma, and erythrocytes, and microscopically-detectable fatty vacuolization of the adrenal zona fasciculata. The principle effect in females at this dose was depression of brain, plasma, and erythrocyte ChE.

At the 5 mg/kg bw/day treatment level, the effects were similar but less pronounced in both male and female rats, however the body weight differences were not apparent. The only effect still present in either males or females at 1 mg/kg bw/day was depression of plasma and erythrocyte ChE but not brain ChE. There were no biologically significant effects of treatment at the 0.1 mg/kg bw/day dose level.