Chlorpyrifos

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

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:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

no

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: AGR 219646
Purity: 100.0 ± 0.9%

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 Breeding Laboratories, Inc., Kingston, New York
- Age at study initiation: Approximately 13 weeks
- Weight at study initiation: Male: 249.4-251.1 g; Female: 142.9-145.3 g
- Housing: When the animals were not in exposure chambers, they were pair-housed in stainless steel cages with wire bottoms in a room designed to maintain adequate environmental conditions concerning temperature and humidity, and were regulated for the specific species under test.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Approximately 3 weeks. The animals were additionally acclimated to periodic confinement in the nose-only tubes over a 4 week period.

DETAILS OF FOOD AND WATER QUALITY: Analysis of the feed was performed to confirm that the diet provided adequate nutrition and to quantify the levels of selected contaminants associated with the formulation process. Analysis of the tap water was also periodically performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 50

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 44 liter ADG-design nose-only chambers
- Method of holding animals in test chamber: The animals were placed in the exposure ports and held by nose-only tubes
- Source and rate of air: The test atmospheres were generated by passing warmed air (<40°C) through glass pipes containing glass beads coated with the test substance.
- Method of conditioning air: The chambers were operated for approximately 0.5 hours before placing the animals in the exposure ports to more rapidly attain the targeted exposure concentrations.

TEST ATMOSPHERE
- Brief description of analytical method used: The test substance was quantitated with a Varian 3700 gas chromatograph coupled to a Hewlett-Packard 3350 laboratory automation system for integration of peak areas.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance concentrations were typically determined three times/chamber/day except the control chamber, which was analyzed weekly. Sampling intervals typically covered 105 min/sample to trap quantifiable amounts of test substance and to allow a valid TWA assessment. A gas chromatographic method was developed for the analysis of test substance vapor in this study. Automated sampling of the chamber air via solenoid valves was not possible due to adsorption of test substance to surfaces and to the very low chamber concentrations. A toluene-filled impinge was therefore used to extract and concentrate the test substance prior to analysis. The test substance was quantitated with a Varian 3700 gas chromatograph coupled to a Hewlett-Packard 3350 laboratory automation system for integration of peak areas. The GLC conditions were as follows: column- 6’ x 2 mm id borosilicate glass column packed with 3% OV-1 on 80/100 m chromosorb, oven 220°C, injector 250°C, carrier (nitrogen) flow 20 mL/min, electron capture detector temperature 300°C. Duplicate injections were routinely made of each sample with a Varion 8000 auto-injector. A standard curve was run daily. Chamber distribution studies verified that exposure concentrations varied less than 15% (calculated on a worst-case basis by: range x 100/lowest value) between sampling and selected animal exposure ports.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5.2 other: ppb

Remarks:

75 µg/m3

Doses / concentrations 2

Dose / conc.:

10.3 other: ppb

Remarks:

147 µg/m3

Doses / concentrations 3

Dose / conc.:

20.6 other: ppb

Remarks:

296 µg/m3 (highest attainable concentration)

No. of animals per sex per dose:

10

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: All animals were observed after each exposure period for evidence of treatment-related effects.

BODY WEIGHT:
- Time schedule for examinations: All animals were weighed prior to the first exposure and approximately weekly thereafter.

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to sacrifice
- Anaesthetic used for blood collection: Yes
- Animals fasted: Not specified
- How many animals: All animals

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At necropsy
- Animals fasted: Not specified

URINALYSIS:
- Time schedule for collection of urine: Prior to necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

OTHER: In addition to the routine serum clinical laboratory measurements, cholinesterase activities in plasma, red blood cell (RBC), and brain homogenates were determined. Blood was obtained from the rats by orbital sinus puncture and brain samples were collected at necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: At least four consecutive exposures to the test substance were conducted prior to necropsy. All surviving animals were sacrificed the day following the last exposure. Rats were fasted overnight prior to the scheduled sacrifice. Animals were weighed, anesthetized with methoxyflurane, and their tracheas were clamped prior to decapitation. Weights of brain, lungs, liver, kidneys, adrenals, and testes were recorded from each animal at the scheduled sacrifice. All animals were examined for gross pathological alterations by a veterinary pathologist. The necropsy included in situ examination of the eyes by a glass slide technique with fluorescent illumination. A complete set of tissues were collected from each animal and preserved in neutral, phosphate buffered 10% formalin. The lungs were infused with buffered formalin to their approximate normal inspiratory volume and the nasal cavity was flushed with formalin via the pharyngeal duct to insure rapid fixation of the tissue.

HISTOPATHOLOGY: Histopathologic examinations were performed on complete sets of tissues from all rats in the control and highest exposure groups. Tissues examined histologically were processed by conventional techniques, stained with hematoxylin and eosin, and evaluated by light microscopy.

Statistics:

Descriptive statistics (means and standard deviations) were reported for white blood cell differential counts. Body weights, absolute and relative organ weights, clinical chemistry data, appropriate hematology data, cholinesterase activities, and urinary specific gravities were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett’s test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed respectively by Dunnett's test or Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Statistical outliers were identified by a sequential test but were not excluded from analyses.

Since multiple, interrelated parameters were statistically compared in the same group of animals, the frequency of false positive errors may have been much greater than the nominal alpha level. Thus, in addition to statistical analyses, the final toxicologic interpretation of the data also considered factors such as dose-response relationships and whether or not the findings appeared to be plausible and consistent in the light of other biologic findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the first month of the study rats in all exposure groups, including controls, had slight red staining around the eyes and nares, probably due to the stress associated with repeated confinement in the exposure tubes. This stress response did not occur in the final 2 months of the study. No other abnormalities were observed in any other animals during the daily observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat in the control group died during the course of the study. Based on gross and histopathologic observations, this animal apparently died from suffocation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although there were no significant differences from controls in group mean body weights of any exposure group, rats in all groups (including controls) did not gain as much weight as rats that are not subjected to daily restraint for nose-only exposures.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no alterations in hematologic parameters in the male rats. In female rats, the only hematologic parameter that was statistically different from controls was a slight decrease (<4%) in erythrocyte counts at all exposure levels. These differences lacked a dose-response relationship and there were no microscopic changes in the peripheral blood or bone marrow sections suggestive of hematologic dysfunction. In addition, the red blood cell counts were within the range of our laboratory's historical control data. These differences were, therefore, not considered to be due to exposure to the test substance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum urea nitrogen was elevated (~13%) in males at all exposure levels, although only the low and high exposure groups were statistically different from controls. There was no corresponding increase in serum electrolytes or histopathologic evidence of renal toxicity. In addition, the values for serum urea nitrogen were within the range of our historical control data. These differences were, therefore, not considered to be due to exposure to the test substance. The slight increase (<2%) in serum sodium in low dose females was not dose-related and was too small to be biologically significant.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related effects observed in urinalysis data.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant differences from controls in the mean terminal fasted body weights or in absolute and relative organ weights of any exposure group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related changes observed in tissues examined either grossly or histologically.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No differences from controls were noted in either plasma, red blood cell or brain cholinesterase activities.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related changes observed in tissues examined either grossly or histologically.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

> 287 other: µg/m3

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest attainable concentration

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOEL (rat): >20.6 ppb (287 µg/m3), highest attainable concentration

Executive summary:

The study was conducted following the guideline EPA OPP 82-4. Fischer 344 rats (10/sex/exposure concentration) were exposed nose-only to 0. 5.2. 10.3 or 20.6 ppb (0. 75. 147 or 296 µg/m3) test substance for 6 hrs/day, 5 days/week for 13 weeks. The exposure concentrations were limited by the low vapor pressure of test substance (theoretical maximum vapor concentration of 25 ppb at 25°C). No treatment-related signs of toxicity or changes in body weights were detected during the course of the study. Urinalysis, hematology, clinical chemistry, organ weight. gross pathologic and histopathologic evaluations were performed at the end of the study with no treatment-related effects observed. In addition, no differences from controls were noted in either plasma, red blood cell or brain cholinesterase activities. The results of this study indicate that the no-observed-effect level (NOEL) for the test substance was greater than the highest attainable concentration, 20.6 ppb, in male and female Fischer 344 rats.