Chlorpyrifos

Endpoint summary

Description of key information

Study Type	Species	Findings	Guideline	Reliability
Oral 4-week Feed	Mice	MTD: 15 ppm (corresponding to dose levels of 2.7 and 3.4 mg/kg body weight/day for males and females)	no guideline	2
Oral 13-week Feed	Rat	NOAEL 0.1 mg/kg/d; LOAEL 1 mg/kg/d (RBC cholinesterase inhibition	EPA OPP 82-1	1
Oral 13-week capsules	Dog	NOEL 0.1 mg/kg/d (based on body weight and weight gain; neuropathology; histopathology: non-neoplastic	No guideline	2
Oral 24 months Feed	Rat	NOEL: 0.1 mg/kg/d (based on body weight and weight gain; urinalysis; organ weights and organ / body weight ratios; histopathology: non-neoplastic; histopathology: neoplastic; Cholinesterase activity)	EPA OPP 83-1, OPP 83-2	1
Oral 105 weeks Feed	Mouse	NOAEL: >15 ppm (results of this study revealed neither evidence of toxicity nor an oncogenic response in mice administered test substance in the diet during the major portion of their natural lifespan)	No guideline	2
Oral 6 weeks Feed	Dog	NOAEL: 1 mg/kg/d; no effects observed at highest dose tested	No guideline	2
Oral 2 years Feed	Dog	LOEL: 1 mg/kg/d (based on ChE depression in red cells)	No guideline	2
13 weeks Inhalation	Rat	NOEL: >287 µg/m3 (highest attainable concentration)	EPA OPP 82-4	1
2 weeks Inhalation	Rat	NOAEL: >12 ppb (Due to its low maximum attainable vapor concentration, the test substance vapor exposure appears to have a low potential for sub-chronic inhalation toxicity in rats)	No guideline	2
2 weeks Inhalation	Rat	NOAEL: >5 ppb (No effects based on daily clinical observations, body weights, or gross pathology in a 2 week study in rats)	No guideline	2
21-Day Dermal	Rat	NOAEL: > 5 mg/kg/d (highest level tested)	EPA OPP 82-2	1

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: 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 OPP 83-1 (Chronic Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-2

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot Number: AGR 214637
Purity: 98.5%

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

The Fischer-344 rat was selected as the experimental model because it has been used extensi vely for toxicity-oncogenicity studies, has a well known biological database and a high degree of uniformity, and is available in large numbers from a reputable supplier. In addition, this strain of rat was used in the subchronic study.

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

and diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to confirm test material concentrations were performed on samples of premixes, control feed, and all dose-level diets at the start of the study and at approximately 3-month intervals until the end of the study. Overall, the analytically determined dietary concentrations were sufficiently comparable to targeted concentrations for meaningful interpretation of the study.

Duration of treatment / exposure:

24 months

Frequency of treatment:

Daily

Dose / conc.:

0.05 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

60 (10 rats/sex/dose level were randomly designated at the start of the study for an interim sacrifice at 12 months. The remaining animals were continued on test until 24 months or they died or were killed moribund)

Control animals:

yes, plain diet

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs for individual animals which suggest a relationship to test substance exposure. In addition to the individual clinical observations, a group observation of mild yellowish perineal staining was noted for the top dose female rats for much of the study. 30-40% of the 10 mg/kg bw/day group females had perineal staining from approximately the 6th month until it was no longer distinguishable at about 20 months. A significant amount of perineal staining was not present in high dose males or any other group of animals.
There is no indication in these data of any excess of masses, unusual tumors, or early onset in any treatment group versus respective controls. The type and incidence of cutaneous and subcutaneous "masses" noted were within the normal range of neoplasms for the Fischer-344 rat.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no statistically or biologically significant differences in the mortality pattern between controls and any treated dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, the body weights of rats in the 1 and 10 mg/kg bw/day groups were consistently lower than the control group in a dose-related manner, throughout the study. At the higher dose level, the weight depression was approximately 7-9% throughout the study and was consistently identified as statistically significant. While statistically significant at most measurement intervals, the weight gain depression in the 1 mg/kg bw/day group was less pronounced than in the higher dose level. The group mean body weights of females in the top dose level were slightly but statistically significantly decreased relative to controls from approximately the second month on study through most of the first year. After that time, they were not distinguishable from control values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no appreciable difference in the amount of feed consumed by any treatment group relative to controls.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the findings in treated animals were interpreted as direct effects of exposure to test substance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were a number of values which were statistically identified as different from the control means at various dose levels and sampling periods. However, in male rats the only parameters which were consistently different and appeared to be associated with the treatment regimen were decreases in cholesterol, total protein and globulin in the 10 mg/kg bw/day group at 6, 12, and 18 months. (These differences were not remarkable at 24 months; however, this is not unexpected due to the high variability and intercurrent disease entities in the geriatric population). The decreases in these parameters do not appear to indicate any direct organ toxicity but are probably secondary effects of altered metabolism, possibly as a result of the adrenal changes.

As in males, the cholesterol values for female rats in the top dose were significantly decreased at 6, 12, and 18 months. Globulin values were significantly decreased at 6 and 12 months but not at 18 or 24 months. All other statistically identified values in males and females for clinical chemistry or electrolytes were considered incidental and due to random variability because they were either not dose-related or were inconsistent over time as to occurrence or direction of change relative to control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine specific gravity was consistently elevated in male rats of the top dose group at all sampling periods and was statistically significant at all but the 12-month time period. While this persistent elevation appears to be treatment related, it does not appear to be an indicator of a direct renal effect since there was significantly less renal pathology in the top dose than in the control animals.
Female rats in the high dose also had a mild but statistically significant increase in urine specific gravity at the 6- and 12-month sampling periods, but not after 12 months. All other urinary parameters in either males or females appeared to be unaffected by test substance administration.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At both sacrifice intervals, the body weights of the top dose males were lower than controls, indicative of the decreased weight gain throughout the study in this dose group. The smaller size of the male rats of the 1 mg/kg bw/day group is not apparent in the sample chosen for the 12-month sacrifice. At the terminal sacrifice, the final fasted body weight of the 0.1 mg/kg bw/day group is statistically significantly decreased relative to controls, which probably represents group-to-group variability in the geriatric population. The in-life body weights of this group were comparable to controls throughout the entire study.
The only organ weight changes in males which appear to be effects of test compound administration are the significant increases in absolute and relative adrenal weights of the top dose animals at both sacrifice intervals. The other dose levels were unaffected. The statistically identified increase in relative brain weight at both necropsies and the decreased absolute kidney and liver weights at 24 months in the top dose males were considered a reflection of the lower body weight in this group of animals.
In female rats, there were no biologically significant changes in body or organ weights at the 12-month sacrifice interval. At 24 months; however, the relative and absolute adrenal weights in the top dose animals were significantly increased similar to the male rats but to a lesser degree. The absolute brain weight, but not the relative weight, in female rats of the 10 mg/kg bw/day group was statistically significantly increased at the 24-month sacrifice. With the greater variability at this age, this increase cannot be causally related to test substance administration.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious pattern of changes in any dose group at either sacrifice interval suggestive of a treatment-related effect.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, plasma ChE was consistently depressed to a statistically significant degree at each of the sampling periods in the 1 and 10 mg/kg bw/day dose groups. Male erythrocyte ChE was also consistently less than control values in the 1 and 10 mg/kg bw/day groups, although not statistically significant at either dose level at 12 months. The depression in RBC ChE was considered an effect of treatment at both dose levels. Brain ChE was only measured at 12 and 24 months, and biologically significant depression was present only at the top dose level at both sampling periods. At the 12-month sampling period, all four treatment groups were statistically different from controls but only the difference at the highest dose was considered an effect of exposure to test substance for the following reasons: 1) the ChE values for the 3 lower dose groups were virtually identical over a 20-fold range of dose levels (no dose response), 2) there was no indication at the 24-month sacrifice of any effect over the same dose range with a larger sample size (20 vs 10), and no depression of brain ChE was present after 13 weeks at 1 mg/kg bw/day or lower. It is unclear why the three lower dose groups were "statistically" different from the control group at 12 months, but it is clear from examination of the data, in context with available data from this and other studies, that the biologic effect of test substance-induced inhibition of brain ChE occurs between 1 and 10 mg/kg bw/day.

In females, there was also a consistent, statistically significant depression of plasma ChE at 1 and 10 mg/kg bw/day. A slight but statistically significant decrease in plasma ChE relative to controls was present in the female 0.1 mg/kg bw/day dose group at 12 and 18 months, but not at 6 and 24 months. Due to the lack of repeatibility and the small magnitude of the statistically significant differences (13-14%), it cannot reasonably be concluded that these differences were due to test compound administration. Erythrocyte ChE values in females were comparable to controls for all dose levels and all time periods with the exception of a statistically identified decrease in the top dose at the the 12-month sampling period. Although erythrocyte ChE depression has been demonstrated in female rats at levels as low as 1 mg/kg bw/day in the 13-week study, there was insufficient evidence in the present study to conclude that administration of test substance had any effect on the cholinesterase levels of erythrocytes under the conditions of the test.

Brain ChE was significantly depressed at the 10 mg/kg bw/day level in females at both the 12 and 24 month sampling period. The value for brain ChE in females at the 1 mg/kg bw/day was statistically identified as different from the control value; however, this difference was not interpreted as an effect of the test material. There was less than a 5% difference in the control and the treated value in this instance (11.75 vs 11.20), and the two groups were considered indistinguishable from a biologic standpoint.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the 12-month sacrifice, the only histopathologic alteration associated with test substance administration was an excess of fatty vacuolization of the zona fasciculata in the adrenal cortex of male rats given 10 mg/kg bw/day of the test material. This change would appear to account for the increase in adrenal weight noted for this group of male rats at this sacrifice interval. As noted in the data, control male rats of this age normally have some degree of mild vacuolization of the mid-adrenal cortex (recorded as very slight), but females do not. The alteration in top dose males was an exacerbation of this vacuolization without obvious concommitant necrosis or cellular degeneration. There were no histopathologic changes in female rats at any dose level associated with the test material at 12 months.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic examination of male rats of the top dose designated for terminal sacrifice revealed changes in three organ systems that appear to be treatment-related: an increase in slight adrenal cortical vacuolization similar to that seen at the 12-month sacrifice; a decrease in the severity of chronic progressive glomerulonephropathy (chronic renal disease); and a decreased incidence of mild biliary hyperplasia in the liver. In female top dose rats, the only apparent treatment-related effect was a similar decrease in the incidence of biliary hyperplasia. There were no histopathologic changes in any dose level other than the 10 mg/kg bw/day level which were interpreted as effects of test substance administration. There were no statistically identified linear trends or pairwise increases in any tumors for either males or females at any dose level. In addition, the total number of primary, benign, or malignant tumors was comparable in all groups.

Key result

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: neoplastic

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

other: Cholinesterase activity

Key result

Lowest effective dose / conc.:

1 mg/kg bw/day (nominal)

Organ:

other: Cholinesterase activity

Conclusions:

NOEL (rat): 0.1 mg/kg bw/day

Executive summary:

The study was conducted following the guideline, EPA OPP 83-1 and 83-2. Sixty rats/sex/dose level were fed test substance in their diets at concentrations formulated to provide 0 (control), 0.05, 0.1, 1, or 10 mg/kg bw/day. Ten rats/sex/dose level were randomly designated at the start of the study for an interim sacrifice at 12 months. The remaining animals were continued on test until 24 months or they died or were killed moribund. Parameters measured during the study were clinical observations; body weights; feed consumption; hematology; clinical chemistries and electrolytes; plasm, RBC and brain cholinesterase; urinalyses; and gross and histopathologic examination.

The primary effects of administration of 10 mg/kg bw/day to male rats for up to 2 years were a decrease in body weight gain relative to controls, depression of plasma, RBC, and brain cholinesterase, and an increase in the size of the adrenal glands characterized by increased fatty vacuolization of the zona fasciculata. Other effects of the treatment regimen, which may be secondary to these changes, included decreases in serum cholesterol, total protein and globulin, an increase in urine specific gravity, and a decrease in some common geriatric conditions such as chronic renal disease and biliary hyperplasia. The mechanism of these secondary effects is unknown, but they may be the result of changes in adrenal function, metabolic alterations associated with the smaller size of the animals, and/or the results of mild chronic stress.

Altered parameters in females at the top dose were generally similar to those in males but were less pronounced. There was a transient depression of body weight and an increase in adrenal weight at the terminal sacrifice, however, there was no histopathologic lesion associated with this increase in adrenal weight. Plasma and brain cholinesterase values were depressed but not erythrocyte levels. Like males, serum cholesterol and globulins were mildly depressed, and urine specific gravity was elevated, generally coinciding with the periods of weight gain depression, and there was a decreased incidence of biliary hyperplasia. Clinically, there was also mild perineal staining in these female rats throughout much of the study.

At the 1 mg/kg bw/day treatment level, the only effects of treatment in males were depression of body weight gain and a depression of plasma and RBC cholinesterase levels. In females, plasma cholinesterase depression was the only effect at this dose level.

The chronic toxicity noted at the top dose in this study was considered indicative of a sufficient challenge for the assessment of oncogenicity without producing excessive mortality. There was, however, no increase in tumors of any type in any organ or tissue at any of the dose levels tested.

The No-Observed-Effect-Level (NOEL) for all parameters in both males and females for this study was 0.1 mg/kg bw/day.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot Number: MM820905-610
Purity: 95.7%

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

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

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

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.

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.

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.

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

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

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

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.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was conducted to evaluate the long-term toxicity and oncogenic potential of the test substance which might result from lifetime ingestion of the test substance in the diet of mice. Groups of CD-1 mice (56/sex/group) were maintained on diets containing 0, 0.5, 5 and 15 ppm of test substance for 105 weeks. Parameters evaluated included behavior, physical condition, mortality, food consumption, body weights, organ weights and tumor incidence including gross and microscopic examination of all mice which died, or were killed in a moribund state or sacrificed at termination of the study.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Ref. No. 1-550-2
Purity: 99.6 ± 0.9%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

Daily

Dose / conc.:

0.5 ppm

Remarks:

0.05 mg/kg/day

Dose / conc.:

5 ppm

Remarks:

0.5 mg/kg/day

Dose / conc.:

15 ppm

Remarks:

1.5 mg/kg/day

No. of animals per sex per dose:

56

Control animals:

yes, plain diet

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in behavior or physical appearance during the course of the study that could be attributed to ingestion of test substance in the diet.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In neither sex was there a statistically significant difference between the control and treated groups in survival rate.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences were observed between the mean body weights of the control and treated groups of animals during the course of the study. The mean body weight of males ingesting 15 ppm test substance showed a slight decrease at day 420 when compared to the mean values of the control and other treatment groups; at each of the other observation intervals their mean body weight was comparable to other male groups. This isolated observation was considered of no toxicologic significance. The mean body weight of all treated groups of female mice was greater than the control mean beginning with day 84 of the study. All male and female study groups, including controls, showed a slight decrease in mean body weight during the final two months of the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreases in mean daily food consumption were observed on study day 168 in males given 5 ppm and on study days 504 and 588 in males given 0.5 ppm. In female mice, a statistically significant decrease in food consumption occurred on study day 476 in females given 5 ppm and on study day 504 in females given 15 ppm (it should be noted that the mean food consumption of control females showed a disproportionate increase at these same intervals). Food consumption of female mice given 0.5 ppm showed a statistically significant increase over the mean control value on day 28. The food consumption curves show that food consumption by all groups of male and female mice, including controls, showed a decline from 2 to 3 months until 10 to 11 months when it was once again increased. Thereafter all study groups showed generally consistent deviations in mean food consumption. In summary, the few sporadic instances in which there was a statistical decrease or increase in food consumption between the control and various treatment groups showed no consistent trend and were, therefore, considered of no toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights were unaffected at any dose level of the test substance. The differences in heart and kidney weights of males of the low dose level were not considered to be of toxicological significance due to the absence of a dose-response relationship and the lack of gross or microscopic lesions unique to this group as compared to other male groups including the controls. Similarly, the statistical difference in the weight of the liver of females of the low and intermediate dose levels were not considered to be of toxicological significance, particularly in the absence of a dose response relationship.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the major and minor lesions or the other gross and microscopic non-neoplastic lesions were considered related to lifetime ingestion of test substance in the diet at any of the dose levels tested.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A variety of non-tumor lesions, categorically of a degenerative, inflammatory or proliferative nature was observed in a random fashion in animals of both the treated and control groups. Although the incidence rate of hyperplastic nodules of the liver of males of the intermediate dose level group was increased statistically over that of control males, the increased incidence was considered fortuitous and unrelated to treatment since it was not observed in a dose-response fashion and also showed a lack of correlation with the incidence rate of hyperplastic nodules in other groups of treated and control animals. Thus, all of the non-tumor lesions were considered to be spontaneous and unrelated to treatment with test substance.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A variety of types of tumors, tumor-like lesions and other non-proliferative lesions occurred in a spontaneous or random fashion in animals of both the control and treated groups. None of these lesions were considered to be related to ingestion of test substance in the diet.

Key result

Remarks on result:

other: results of this study revealed neither evidence of toxicity nor an oncogenic response in mice administered test substance in the diet during the major portion of their natural lifespan.

Key result

Critical effects observed:

no

Conclusions:

Results of this study revealed neither evidence of toxicity nor an oncogenic response in mice administered test substance in the diet during the major portion of their natural lifespan.

Executive summary:

This study was conducted to evaluate the long-term toxicity and oncogenic potential of test substance which might result from lifetime ingestion of test substance in the diet of mice.

Groups of CD-1 mice (56/sex/group) were maintained on diets containing 0, 0.5, 5 and 15 ppm of test substance (approximately 0.05, 0.50 and 1.50 mg/kg/day) for 105 weeks. Parameters evaluated included behavior, physical condition, mortality, food consumption, body weights, organ weights and tumor incidence including gross and microscopic examination of all mice which died, or were killed in a moribund state or sacrificed at termination of the study.

None of the above parameters appeared to be affected by treatment at any dose level of test substance. A variety of types of tumors, tumor-like lesions and other non-proliferative lesions occurred in a spontaneous or random fashion in animals of both the control and treated groups. None of these lesions were considered to be related to ingestion of test substance in the diet. Thus, results of this study revealed neither evidence of toxicity nor an oncogenic response in mice administered test substance in the diet during the major portion of their natural lifespan.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female CD-1 mice were provided diets containing 0 or 15 ppm of test substance for up to four weeks for the purpose of evaluating whether a dietary concentration of 15 ppm was associated with any significant biologic response in mice.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Lot Number: MM820905-610
Purity: 95.7%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

and diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Dose / conc.:

15 ppm

Remarks:

corresponding to dose levels of 2.7 and 3.4 mg/kg body weight/day for males and females, respectively

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight retardation of body weight gain was observed among the male mice treated for four weeks relative to their control counterparts. These treated mice showed reduced weight gains at each weekly interval. On occasion, differences from the control values were statistically significant. At the termination of the study, the treated males showed a 25% reduction of body weight gain relative to controls. Growth of the treated female mice was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on the feed consumption of mice of either sex. The male animals showed a slight reduction in test substance intake (expressed as mg/kg body weight/day) as the study progressed; a similar trend was not apparent among the treated females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant depression of cholinesterase activity in plasma and erythrocytes was noted among treated mice of both sexes at each sampling time. The greatest response occurred in plasma enzyme activity which was reduced in treated animals by 88-91% relative to controls; erythrocyte cholinesterase activity was depressed by 40-53% compared to control values. Differences in sensitivity between the sexes were not apparent. The effects on the plasma and erythrocyte enzyme activities were judged to be treatment-related in view of the recognized sensitivity of cholinesterases to inhibition by organophosphates.

A statistically significant reduction of brain cholinesterase activity was noted for the treated male mice sacrificed at four weeks. In contrast, the treated females killed at the termination of the study showed a statistically significant increase in brain cholinesterase activity. Given the small magnitude of the changes and the lack of consistency of response between sexes, these findings were considered to be of questionable toxicological significance. Treated animals of both sexes sacrificed at one week showed no differences from controls in brain cholinesterase activity.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute brain weights of the treated male mice killed after one week were statistically identified as being lower than corresponding control values. The effect was judged to reflect normal variability since the corresponding relative brain weights (g/100 g body weight) were unaffected and comparable changes were not evident among the treated females sacrificed at one week or among treated animals of both sexes killed after four weeks. Absolute and relative weights of heart, liver, kidneys and testes measured at the termination of the study were comparable among the control and treated groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Observations made during the gross pathological examination of all mice at four weeks were non-remarkable. All morphologic lesions encountered were judged to reflect normal background lesions and were judged not to be associated with treatment.

Dose descriptor:

other: MTD

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to dose levels of 2.7 and 3.4 mg/kg body weight/day for males and females, respectively

Conclusions:

Maximum tolerated dose (mice): 15 ppm

Executive summary:

Male and female CD-1 mice were provided diets containing 0 or 15 ppm of test substance (corresponding to dose levels of 2.7 and 3.4 mg/kg body weight/day for males and females, respectively) for up to four weeks for the purpose of evaluating whether a dietary concentration of 15 ppm was associated with any significant biologic response in mice.

Groups of male and female mice (40/sex/group) were randomly allotted into two sub-populations (20/sex/group) which were maintained on the diets for one or four weeks. Parameters evaluated were in-life observations, body weight changes, feed consumption, gross examination at necropsy (4 weeks only), organ weights and cholinesterase activities in plasma, erythrocytes and brain.

Administration of the diet containing 15 ppm of chlorpyrifos induced significant depressions of plasma and erythrocyte cholinesterase activities among both sexes at one and four weeks. Plasma enzyme activity was reduced by 88-91% relative to control values; erythrocyte cholinesterase activity was depressed by 40-53%. Body weight gains were identified to be statistically significantly decreased in male mice sacrificed after four weeks of dietary intake of 15 ppm test substance. All other parameters measured were unaffected by treatment. The significant reduction in cholinesterase activities of plasma and erythrocytes in both sexes, together with the statistically significant retardation of body weight gain of the treated male mice indicated that ingestion of 15 ppm test substance in the diet was adequate to elicit significant biologic responses. Therefore, a dietary intake of 15 ppm test substance was considered to be representative of the maximum-tolerated dose for this strain of mouse.

Reference 5

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

DOWCO 179 was administered to male and female beagle dogs in their diets at doses of 0, 3.0, 1.0, 0.1, 0.03 and 0.01 mg/kg/day for periods up to two years. Parameters evaluated were appearance and demeanor; body weight; food consumption; hematology; urinalyses; plasma, red cell, and brain cholinesterase (ChE); clinical chemistry--blood urea nitrogen, alkaline phosphatase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and bromsulfalein retention; and pathological studies after one year on experiment, after three months on control feed subsequent to receiving the test diets for one year, and after two years on experiment—these included evaluation of organ weights and gross and histological examination of organs and tissues.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos
Lot No. CPS23-CD235 C
Purity: 98.8%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

acetone

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.01 mg/kg bw/day (nominal)

Dose / conc.:

0.03 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Dogs were observed daily.

Body weights - Recorded weekly during months 1-6 and biweekly thereafter.

Food consumption - Recorded weekly during months 1-3 and 1 week/month thereafter.

Hematologic studies - Conducted on dogs at the 0, 3.0 and 1.0 mg/kg/day doses twice pre-test and at 1, 6, 12, 18 and 24 months.

Urinalyses - Conducted on dogs at the 0, 3.0 and 1.0 mg/kg/day doses once pre-test and after 1 month, one year (approximately), and two years.

Other clinical chemistry studies - BUN, AP, SGOT and SGPT were measured on all dogs twice pretest and after 1 and 24 months on the test diets. Dogs receiving 0, 3.0 and 1.0 mg/kg/day DOWCO 179 were also sampled for these determinations after 6, 12 and 18 months on test. Bromsulfalein (BSP) retention was measured on all dogs twice pretest and terminally, and on the dogs receiving 0, 3.0 and 1.0 mg/kg/day DOWCO 179 after 12 months.

Sacrifice and pathology:

Physical examination - All dogs were given complete physical examinations prior to termination. These included routine neurologic and ophthalmoscopic evaluations.

Pathology - All dogs were sacrificed and necropsied after two years on the test diets. Histopathological examination of tissues was conducted on dogs receiving 0 and 3.0 mg/kg/day DOWCO 179 only.

Other examinations:

Cholinesterase activity - Measured in plasma and red cells from all dogs twice pre-test and after being maintained for 1 week and 1, 3, 6, 12, 15, 18 and 24 months on the test diets.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs of toxicity were observed in any of the dogs in either phase of the experiment. Greater than normal cholinergic activity - excessive salivation, miosis, diarrhea - were not observed at any time.

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

No trends were noted which suggest a compound-related effect on the various hematologic parameters. This is true either when pretest
and post-test values for the same animals are compared or when the values of the treated and control group are compared.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only noteworthy change was an increased mean liver/body weight ratio in the male dogs receiving 3.0 mg/kg/day DOWCO 179.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

There were statistically significant decreases in the plasma ChE activity of dogs, male and female, receiving 0.03 mg/kg/day DOWCO 179 at some of the sampling times over the one year test period. The average plasma ChE activity of the males receiving this dose over this duration was 82.4% of the average pretest value. For the females it was 82.7%. Therefore, the depression of plasma ChE activity in the male and female
dogs receiving 0.03 mg/kg/day DOWCO 179 is well within the range generally accepted as physiologically insignificant. Plasma ChE activity of male and female dogs receiving 0.01 mg/kg/day DOWCO 179 was not significantly depressed. The ChE activity of plasma of all dogs showing a significant depression associated with the ingestion of diets containing DOWCO 179 for one year returned to pretest levels within two weeks after being fed control diet.

ChE activities of red cells of male and female dogs receiving 3.0 and 1.0 mg/kg/day DOWCO 179 were depressed. No significant depression occurred in either sex receiving 0.1 mg/kg/day and below for one year. Red cell ChE activity returned to pretest levels in male and female dogs maintained on control feed for three months subsequent to receiving doses of 3.0 and 1.0 mg/kg/day DOWCO 179 for one year. ChE activity in samples of brain were similar for control and all groups of male and female dogs receiving DOWCO 179.

Phase B - ChE activities in plasma of male and female dogs receiving 3.0, 1.0 and 0.1 mgjkgjday DOWCO 179 were significantly depressed. In males receiving 0.03 mgjkgjday, the plasma ChE activity was statistically significantly decreased at some of the samplings. However,
the mean activity found for all of the samplings was 101% of the pretest mean and 76% of the fuean untreated control. For females receiving this dose, corresponding figures were 115% and 90% respectively. No statistically significant decreases in plasma ChE activity were found
in either sex receiving 0.01 mgjkgjday. ChE activity of red cells was depressed only in male and female dogs receiving 3.0 and 1.0 mgjkgjday DOWCO 179. Although the red cell ChE activity of females receiving 0.1 mgjkgjday appears lower than controls at the various test periods (two year mean of 73% of control), this group's mean was also lower than the control group at the pretest samplings (79% of control mean) .

Brain ChE activities appear to be slightly depressed in both sexes receiving the 3.0 mgjkgjday dose for two years These decreases, however, were not statistically significant when compared with controls. ChE activities in the brain of dogs receiving lower doses were within the range of controls.

Key result

Dose descriptor:

LOEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: ChE depression in red cells

Conclusions:

No significant effects were associated with the ingestion of 0.03 or 0.01 mg/kg/day DOWCO 179 by dogs for two years. At higher doses, up to 3.0 mg/kg/day, varying degrees of ChE depression occurred without accompanying evidence of toxicity.

Executive summary:

DOWCO 179 was administered to male and female beagle dogs in their diets at doses of 0, 3.0, 1.0, 0.1, 0.03 and 0.01 mg/kg/day for periods up to two years. Parameters evaluated were appearance and demeanor; body weight; food consumption; hematology; urinalyses; plasma, red cell, and brain cholinesterase (ChE); clinical chemistry--blood urea nitrogen, alkaline phosphatase, serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, and bromsulfalein retention; and pathological studies after one year on experiment, after three months on control feed subsequent to receiving the test diets for one year, and after two years on experiment—these included evaluation of organ weights and gross and histological examination of organs and tissues.

 

No significant toxicological effects were detected in any of the dogs receiving the diets containing DOWCO 179. ChE depression was noted in plasma from male and female dogs at doses of 3.0, 1.0 and 0.1 mg/kg/day. Although plasma ChE in dogs receiving 0.03 mg/kg/day showed statistically significant decreases at some of the sampling times, the overall mean plasma ChE values for these dogs were greater than 80% of pretest and/or untreated control means. ChE in red cells was depressed in male and female dogs receiving doses of 3.0 and 1.0 mg/kg/day DOWCO 179 only. Signs associated with greater than normal cholinergic activity were not observed in any dogs throughout the study. When dogs were maintained for one year on diets providing doses of DOWCO 179 which caused a significant depression of plasma and red cell ChE and then were subsequently placed on control diets, the plasma and red cell ChE returned to normal within two weeks and three months, respectively. It is concluded that no significant effects were associated with the ingestion of 0.03 or 0.01 mg/kg/day DOWCO 179 by dogs for two years. At higher doses, up to 3.0 mg/kg/day, varying degrees of ChE depression occurred without accompanying evidence of toxicity.

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The purpose of this study was to characterize the potential histopathologic alterations of the adrenal glands in Beagle dogs following dietary administration of Chlorpyrifos Technical. Male and female Beagle dogs (n = four/sex/dose level; six months old) were fed diets formulated to contain 0,0.5, 1 or 2 mg/kg/day Chlorpyrifos Technical for six weeks. Paraffin embedded, hematoxylin and eosin stained sections of adrenal glands from all dogs were examined microscopically. In addition, frozen sections from the control and 2 mg/kg/day dose groups were stained for lipid with oil red O.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Dursban-F (Chlorpyrifos Technical)
Lot 7299412, TSNlO0759
Purity: 97.6%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

acetone

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

six weeks

Frequency of treatment:

Seven days/week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

0.5 mg/kg bw/day (actual dose received)

Dose / conc.:

1 mg/kg bw/day (actual dose received)

Dose / conc.:

2 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Sacrifice and pathology:

Paraffin embedded, hematoxylin and eosin stained sections of adrenal glands from all dogs were examined microscopically. In addition, frozen sections from the control and 2 mg/kg/day dose groups were stained for lipid with oil red O.

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at highest dose tested

Conclusions:

There were no treatment-related histopathologic alterations of the adrenal glands in males and females at any dose level.

Executive summary:

The purpose of this study was to characterize the potential histopathologic alterations of the adrenal glands in Beagle dogs following dietary administration of Chlorpyrifos Technical. Male and female Beagle dogs (n=four/sex/dose level; six months old) were fed diets formulated to contain 0,0.5, 1 or 2 mg/kg/day Chlorpyrifos Technical for six weeks. Paraffin embedded, hematoxylin and eosin stained sections of adrenal glands from all dogs were examined microscopically.Inaddition, frozen sections from the control and 2 mg/kg/day dose groups were stained for lipid with oil red O. There were no treatment-related histopathologic alterations of the adrenal glands in males and females at any dose level.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered by capsules to beagle dogs at five dosage levels ranging from 0.01 mg/kg/day to 1.00 mg/kg/day for a period of 90 days (only 32 days for the 0.01 mg/kg/day dosage). Cholinesterase activities of the dogs' blood (or brain) were measured by the Frawley modification of the Michel Delta pH electrometric method.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Dursban
Purity: 97.5%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: capsule

Vehicle:

other: Wayne dog meal

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days (only 32 days for 0.01 mg/kg/day)

Frequency of treatment:

Once daily, seven days per week

Dose / conc.:

0.01 mg/kg bw/day (nominal)

Dose / conc.:

0.03 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

0.3 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Two

Control animals:

yes

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The test substance did not cause a significant lowering of the plasma or red blood cell cholinesterase activity in dogs when administered at 0.01 mg/kg/day. When administered at 0.03 mg/kg/day, plasma cholinesterase activities were significantly lowered after four days of treatment, whereas the red blood cell cholinesterase activity levels were not significantly different from that of the control dogs. The test substance levels of 0.1 mg/kg/day caused significantly lower cholinesterase activities in both plasma and red blood cells, but no differences in brain cholinesterase activities at termination of the 90-day study.

The effect of the test substance on cholinesterase activities, both plasma and red blood cells, is reversible; that is, as soon as the dogs are taken off test substance their plasma and red blood cell cholinesterase activities return to the normal baseline levels within two weeks. The test substance cholinesterase activity inhibition is dose related between the levels of 0.03 and 1.0 mg/kg/day.

Key result

Dose descriptor:

NOEL

Effect level:

0.01 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Cholinesterase activity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.03 mg/kg bw/day (nominal)

System:

nervous system

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

NOEL (Dog): 0.01 mg/kg/day

Executive summary:

The test substance was administered by capsules to beagle dogs at five dosage levels ranging from 0.01 mg/kg/day to 1.00 mg/kg/day for a period of 90 days (only 32 days for the 0.01 mg/kg/day dosage). Cholinesterase activities of the dogs' blood (or brain) were measured by the Frawley modification of the Michel Delta pH electrometric method.

The test substance did not cause a significant lowering of the red blood cell or plasma cholinesterase activities in dogs when administered at 0.01 mg/kg/day. When given at 0.03 mg/kg/day, plasma cholinesterase activities, but not red blood cell cholinesterase activities, were less that 70% of their original baseline activities.

The test substance dosages of 0.1 mg/kg/day or greater resulted in significantly lowered cholinesterase activities in both red blood cells and plasma but not differences in the brain cholinesterase activities at the termination of this 90-day study.

The effect of the test substance on cholinesterase activities, both red blood cells and plasma, is reversible at the levels studied (0.01 - 1.00 mg/kg/day). The test substance cholinesterase activity inhibition is dose related between the levels of 0.03 and 1.00 mg/kg/day. The 0.01 mg/kg/day dosage is considered to be a no-effect level in dogs with the 0.03 mg/kg/day level being a border-line level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.1 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Rat (diet): 90-day oral toxicity study, 50 ppm (males and females) equivalent to 3.6 mg/kg bw/day (males) and 4.1 mg/kg bw/day (females) based on decreased body weight, food consumption, and haematological effects at ≥250 ppm.

Rat (diet): 90-day oral toxicity study, 200 ppm (males) and 150 ppm (females) equivalent to 13.6 and 10.0 mg/kg bw/day, respectively based on hematology changes at ≥400 ppm in males and
≥270 ppm in females.

Mouse (diet): 13-week oral toxicity study, 150 ppm in males and 75 ppm in females equivalent to 26.6 and 15.6 mg/kg bw/day, respectively based on haematology changes at ≥300 ppm in males and
≥150 ppm in females.

Dog (diet): 13-week oral toxicity study, 400 ppm (highest dose tested) equivalent to 14.7 mg/kg bw/day for males and 12.5 mg/kg bw/day for females.

Rat (diet): 24 month oral toxicity study, The NOAEL is 100 ppm for males and females (4.8 mg/kg bw/day and 6.3 mg/kg bw/day respectively). This NOAEL was based on decreased weight gain and haematology changes at 400 ppm.

Rat (diet) 35-week: The NOAEL was 300 ppm (approximately 14.4 mg/kg bw/day) for males based on decreased body weight at 600 ppm and above and 100 ppm (approximately 6.4 mg/kg bw/day) for females based on haematology changes at 300 ppm and above.

Mouse (diet) 104-week: The NOAEL in the 104-week feeding study in mice was 50 ppm for both males
and females (8.7 mg/kg bw/day for males and 10.6 mg/kg bw/day for females). This NOAEL was based on increased mortality in 75 ppm and greater males and females.

Mouse (diet) 23-week study: The NOAEL in mice was 100 ppm for males and females (approximately 16.6 mg/kg bw/day and 23.2 mg/kg bw/day, respectively). This NOAEL was based on haematology changes in males and females at ≥300 ppm.

Dog (diet) 2-year: The NOAEL in the 2-year feeding study in dogs was 100 ppm for males and females (3.0 mg/kg bw/day). This NOAEL was based on histopathology changes in the kidney and spleen at 400 ppm and above.

System:

nervous system

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Lot Number: AGR 219646
Purity: 100.0 ± 0.9%

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

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

Dose / conc.:

5.2 other: ppb

Remarks:

75 µg/m3

Dose / conc.:

10.3 other: ppb

Remarks:

147 µg/m3

Dose / conc.:

20.6 other: ppb

Remarks:

296 µg/m3 (highest attainable concentration)

No. of animals per sex per dose:

10

Control animals:

yes

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.

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.

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

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Female rats were exposed to 0 or 12 ppb test substance vapor for 6 hours/day, 5 days/week for 2 weeks. 12 ppb is approximately one-half the maximum theoretically attainable vapor concentration. Nose-only exposures were selected to allow a well-defined inhalation exposure system. Body weights were measured weekly, and all rats were sacrificed on the day after the last exposure (following three consecutive exposure days).

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Lot Number: AGR 219646
Purity: 99.7%

Species:

rat

Strain:

Fischer 344

Sex:

female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days/week for 2 weeks

Dose / conc.:

12 other: ppb

No. of animals per sex per dose:

6

Control animals:

yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related changes in body weights during the course of the study

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure related effects on the measured haematology

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related effects on cholinesterase activities in the test substance exposed rats. There were statistically identified changes in the mean values for AP, AST, GLOB and TP. Ranges for comparable historical controls (2-week inhalation studies, female Fischer 344 rats) are: AP, 74-222 mU/mL; AST, 72-129 mU/mL; GLOB, 1.0-1.9 g/dL and TP, 4.7-5.5 g/dL. Because the mean values observed in the 12 ppb exposed rats (except TP) were within the range of historical control values, they were not considered to be toxicologically significant. The control total protein mean (5.9 g/dL) was slightly higher than recent control means, the statistical decrease in the exposed rats (5.7 g/dL) was not considered to be biologically significant.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure related effects on the measured urinalysis parameters

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related effects on terminal (fasted) body weights nor on absolute or relative organ weights.

Gross pathological findings:

no effects observed

Key result

Remarks on result:

other: Due to its low maximum attainable vapor concentration, the test substance vapor exposure appears to have a low potential for sub-chronic inhalation toxicity in rats.

Critical effects observed:

no

Conclusions:

Low potential for subchronic inhalation toxicity in a 2 week study in rats

Executive summary:

Female rats (6/group) were exposed to 0 or 12 ppb test substance vapor for 6 hours/day, 5 days/week for 2 weeks. 12 ppb is approximately one-half the maximum theoretically attainable vapor concentration. Nose-only exposures were selected to allow a well-defined inhalation exposure system. Body weights were measured weekly, and all rats were sacrificed on the day after the last exposure (following three consecutive exposure days).

Plasma cholinesterase depression is the most sensitive biological indicator of test substance exposure, therefore, histopathology was not included in this range finding study. There were no exposure-related effects based on cholinesterase activity (plasma, red cell or brain), general clinical observations, body weights, hematology, urinalysis or gross pathology. There were no apparent toxicologically significant effects on clinical chemistry values. Due to its low maximum attainable vapor concentration, test substance vapor exposure appears to have a low potential for sub-chronic inhalation toxicity in rats.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female rats were exposed to targeted concentrations of 0, 1, or 5 ppb test substance vapor for 6 hours per day, 5 days per week for two weeks; there were two control groups. Cholinesterase activity in plasma, red blood cells (RBC) and brain was evaluated in samples obtained on the day after the tenth exposure (following three consecutive exposure days).

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Lot Number: AGR 219646
Purity: 100.0 ± 0.1%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for two weeks

Dose / conc.:

5 other: ppb

Dose / conc.:

1 other: ppb

No. of animals per sex per dose:

6

Control animals:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related clinical signs of toxicity during the course of the study

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related changes in body weights during the course of the study

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean plasma and red blood cell cholinesterase activity, when compared to control group A, was reduced for both male and female rats exposed to 5 ppb. When compared to control group B, cholinesterase activity reduction was additionally seen in brains of females exposed to 5 ppb and plasma of females exposed to 1 ppb. The mean cholinesterase activity values of the two control groups were very similar. Since there were no statistically identified differences between the control groups, the two groups were pooled for further comparison with the chlorpyrifos exposed rats. These comparisons revealed exposure-related decreases in mean plasma and red blood cell cholinesterase activity values in male and female rats exposed to 5 ppb. There also appeared to be a slight decrease in mean plasma cholinesterase activity of females exposed to 1 ppb; however, this decrease may reflect normal variability, and was not considered to be biologically significant.

An apparent slight decrease in mean brain cholinesterase activity of 5 ppb exposed female rats was largely the result of a single rat value (109.9 µ/mL) that was considerably less than the group mean. Plasma cholinesterase has been previously shown to be the most sensitive index of test substance exposure; since the plasma cholinesterase activity of rat was not decreased relative to other 5 ppb exposed females (14.6 µ/mL vs 12.9 µ/mL group mean), the apparent decrease in brain cholinesterase activity was probably not exposure-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related effects on terminal (fasted) body weights or on absolute or relative organ weights.

Gross pathological findings:

no effects observed

Key result

Remarks on result:

other: No effects based on daily clinical observations, body weights, or gross pathology in a 2 week study in rats

Critical effects observed:

no

Conclusions:

No effects based on daily clinical observations, body weights, or gross pathology in a 2 week study in rats.

Executive summary:

Male and female rats (6/sex/exposure concentration) were exposed to targeted concentrations of 0, 1, or 5 ppb test substance vapor for 6 hours per day, 5 days per week for two weeks; there were two control groups. Cholinesterase activity in plasma, red blood cells (RBC) and brain was evaluated in samples obtained on the day after the tenth exposure (following three consecutive exposure days).

There were no effects based on daily clinical observations, body weights, or gross pathology. There were exposure-related decreases in the mean plasma and RBC cholinesterase activity of rats exposed to 5 ppb.

These decreases in cholinesterase activity at low exposure concentrations may in part reflect adsorption of test substance on fur with subsequent dermal absorption and/or oral ingestion due to grooming. Since the routes and magnitude of rat whole-body exposure to test substance are ill-defined, this methodology appears to be poorly suited for extrapolation to humans exposed by inhalation.

Reference 4

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

experimental study

Adequacy of study:

other information

Species:

other: Human

Key result

Dose descriptor:

other: Point of Departure

Effect level:

ca. 0.138 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Acetyl Cklinesterase inhibition

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.966 µg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

40

Species:

other: Human

System:

haematopoietic

Organ:

blood

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

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 OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos (DURSBAN R)
Lot AGR 219646
Purity: 99.98%

Species:

rat

Strain:

Fischer 344

Sex:

female

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

21 days

Frequency of treatment:

6 hours/day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Clinical signs, Functional Observational Battery, Cholinesterase (CHE) activity in plasma, red blood cell (RBC), and brain homogenate.

Hematology (hematocrit (HCT), hemoglobin concentration (HGB), erythocyte count (RBC), total leukocyte count (WBC), and platelet count (PLAT). Differential leukocyte counts were obtained from stained blood smears of all rats.

Clinical chemistry: alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), urea nitrogen (UN), alkaline phosphatase activity (AP), glucose (GLUC), creatinine activity (CREAT), total protein (TP), albumin (ALB), globulin (GLOB), cholesterol (CHOL), triglycerides (TRIG), calcium (CALC) and phosphorous (PHOS). Sodium (Na), potassium (K) and chloride (Cl) were also measured.

Urinalysis: bil irubin, glucose, ketones, blood, pH, protein, and urobilinogen. In addition, the color and appearance of the urine were noted and determinations of specific gravity and microsediment were conducted (determinations of specific gravity were done on individual samples; microsediment was done on pooled urine samples from each dose group).

Sacrifice and pathology:

All surviving animals were sacrificed the day following the last application; rats were fasted prior to the sacrifice. Each animal was weighed, anesthetized, the trachea was clamped and the animal was decapitated. Weights of adrena1s, brain, liver, kidneys and testes were recorded for 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 was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. The brain was bissected longitudinally and one half was utilized for cholinesterase determination. 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 ensure rapid fixation of the tissue. The adrenals, brain, kidneys, liver, peripheral nerve (sciatic), skin (from the treated portion of the back and an untreated site from the dorsal portion of the neck), spinal cord (cervical, thoracic, and lumbar areas), and a few selected tissues which had gross pathologic observations were prepared for histopathological examination by standard methods from all controls and rats treated with 5 mg/kg/day. The tissues were stained with hematoxylin and eosin and examined microscopically by a veterinary pathologist. Although tissues weresimilarly prepared from rats treated with 0.1, 0.5 or 1 mg/kg/day, they were not examined microscopically due to a lack of lesions at the highest dose level.

Statistics:

The statistical analysis outlined below was conducted on parameters measured during the 21-day study; no statistical analysis was conducted on data from the 4-day probe. Descriptive statistics (mean and standard deviation) were calculated for white blood cell differential counts and feed consumption data. Body weights,absolute and relative organ weights, cholinesterase activity, clinical chemistry data, urinary gravity and hematology data (except white blood cell differential counts) were evaluated by Bartlett's test for equality of variances followed by a parametric analysis of variance (ANOVA) and Dunnett's test. Statistical outliers were identified by a sequential outlier test. Only feed consumption outliers were routinely excluded from analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red-brown exudate (staining) around the eyes was noted for most female rats (including controls) throughout the course of the study; a few male rats (including controls) exhibited this red-brown stain around eyes on days 2 and 21 of the study. No other clinical observations were noted.

Dermal irritation:

no effects observed

Description (incidence and severity):

There were no indications of skin irritation based on dermal scores.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

All groups of rats lost weight (2-4%) during the first week of the study. During the second week, however, all groups recovered the lost weight and continued to gain until study termination. Mean body weights were comparable between dose groups ,throughout the course of the study. The initial weight loss was probably the result of stress from the increased handling associated with the bandaging technique.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean feed consumption values were comparable between groups over the study period. Feed consumption was lower for all groups during the first week of the study but increased during the last 2 weeks of the study supporting the theory of initial stress associated with acclimation to the application technique.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects observed in routine hematology parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects observed in routine clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis values for chlorpyrifos treated groups of animals were comparable to controls.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Results of the functional observational battery indicate no appreciable difference between dose groups and control groups. Observations of decreased muscle tone noted for two female rats of the 5 mg/kg/day dose group and increased fecal staining for one male rat of the 0.5 mg/kg/day group were considered incidental findings since no treatment-related effects were observed in other parameters (specifically cholinesterase data).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no indications of systemic toxicity based on terminal body weight or organ weight data.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No indication of toxicity was observed during gross pathologic examination of animals treated dermally with chlorpyrifos for 21 days. Likewise, histopathologic examination did not reveal evidence of toxicity. Each of the few observations made at necropsy was present only in a single rat and all observations were considered to be spontaneous changes unrelated ·to treatment. In addition, all histopathologic observations (except for one control female rat) were of a minimal nature and were considered to be typical spontaneous changes normally present in rats of this strain and age. One female control rat had ureteral and urinary bladder calculi with associated hyperplasia and inflammation of the associated tissues and kidney.

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically identified differences in mean values of brain, plasma, or RBC cholinesterase activity for any group treated with chlorpyrifos. While values for 5.0 mg/kg/day male plasma and RBC cholinesterase and female plasma cholinesterase activities were 7, 11 and 17% lower, respectively, than their concurrent control groups, these values were within the range observed for recent historical controls in this laboratory.

Key result

Dose descriptor:

NOAEL

Effect level:

> 5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted after 21 day dermal exposure at highest level tested

Conclusions:

21-day dermal NOEL (female rat) > 5 mg/kg/day
However, application of 10 mg chlorpyrifos/kg body weight/day for 4 days, produced decreases in plasma (45%) and RBC (16%) cholinesterase activity without evidence of toxicity.

Executive summary:

In a probe study, female Fischer 344 rats {4/dose group) were exposed dermally to chlorpyrifos in corn oil solution at dose levels of 0 (corn oil), 1, 10, 100 or 500 mg/kg body weigh~/day, approximately 6 hours/day, for 4 consecutive days. Clinical observations were recorded daily, body weights were recorded on days 1 and 4, plasma and red blood cell (RBC) cholinesterase activities were measured and a gross necropsy conducted on day 5. There were substantial dose-related decreases relative to control in plasma cholinesterase activities (45, 91, or 98%) for animals that received 10, 100 or 500 mg/kg/day, respectively; RBC cholinesterase activities were also decreased in these groups (16, 49 or 75%, respectively). Animals that received 1 mg/kg/day were unaffected by treatment. Decreases in plasma cholinesterase activity as high as 98%

were not asociated with any changes in body weight, in-life clinical signs, or gross

pathologic alterations.

 

Based on the results of the probe study, 5 rats/sex/exposure group were subsequently exposed to a (corn oil), 0.1, 0.5, 1 or 5 mg chlorpyrifos/kg body weight/day in corn oil solution for approximately 6 hours/day, 5 days/week for 3 consecutive weeks. Body weight, feed consumption, in-life clinical observations, clinical laboratory studies, gross pathology and histopathology were evaluated and found to be unaffected. Plasma, RBC and brain cholinesterase activities measured at necropsy were comparable in treated and control groups. In addition, nothing remarkable was observed in a functional observational battery conducted immediately prior to necropsy.

 

Daily dermal application of up to 5 mg chlorpyrifos/kg body weight/day over 21 days (15

applications) was well tolerated by rats with no indication of systemic toxicity and no decrease in cholinesterase activity, the most sensitive indicator of exposure. However, application of 10 mg chlorpyrifos/kg body weight/day for 4 days, produced decreases in plasma (45%) and RBC (16%) cholinesterase activity without evidence of toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

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 OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos (DURSBAN R)
Lot AGR 219646
Purity: 99.98%

Species:

rat

Strain:

Fischer 344

Sex:

female

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

21 days

Frequency of treatment:

6 hours/day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Clinical signs, Functional Observational Battery, Cholinesterase (CHE) activity in plasma, red blood cell (RBC), and brain homogenate.

Hematology (hematocrit (HCT), hemoglobin concentration (HGB), erythocyte count (RBC), total leukocyte count (WBC), and platelet count (PLAT). Differential leukocyte counts were obtained from stained blood smears of all rats.

Clinical chemistry: alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), urea nitrogen (UN), alkaline phosphatase activity (AP), glucose (GLUC), creatinine activity (CREAT), total protein (TP), albumin (ALB), globulin (GLOB), cholesterol (CHOL), triglycerides (TRIG), calcium (CALC) and phosphorous (PHOS). Sodium (Na), potassium (K) and chloride (Cl) were also measured.

Urinalysis: bil irubin, glucose, ketones, blood, pH, protein, and urobilinogen. In addition, the color and appearance of the urine were noted and determinations of specific gravity and microsediment were conducted (determinations of specific gravity were done on individual samples; microsediment was done on pooled urine samples from each dose group).

Sacrifice and pathology:

All surviving animals were sacrificed the day following the last application; rats were fasted prior to the sacrifice. Each animal was weighed, anesthetized, the trachea was clamped and the animal was decapitated. Weights of adrena1s, brain, liver, kidneys and testes were recorded for 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 was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. The brain was bissected longitudinally and one half was utilized for cholinesterase determination. 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 ensure rapid fixation of the tissue. The adrenals, brain, kidneys, liver, peripheral nerve (sciatic), skin (from the treated portion of the back and an untreated site from the dorsal portion of the neck), spinal cord (cervical, thoracic, and lumbar areas), and a few selected tissues which had gross pathologic observations were prepared for histopathological examination by standard methods from all controls and rats treated with 5 mg/kg/day. The tissues were stained with hematoxylin and eosin and examined microscopically by a veterinary pathologist. Although tissues weresimilarly prepared from rats treated with 0.1, 0.5 or 1 mg/kg/day, they were not examined microscopically due to a lack of lesions at the highest dose level.

Statistics:

The statistical analysis outlined below was conducted on parameters measured during the 21-day study; no statistical analysis was conducted on data from the 4-day probe. Descriptive statistics (mean and standard deviation) were calculated for white blood cell differential counts and feed consumption data. Body weights,absolute and relative organ weights, cholinesterase activity, clinical chemistry data, urinary gravity and hematology data (except white blood cell differential counts) were evaluated by Bartlett's test for equality of variances followed by a parametric analysis of variance (ANOVA) and Dunnett's test. Statistical outliers were identified by a sequential outlier test. Only feed consumption outliers were routinely excluded from analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red-brown exudate (staining) around the eyes was noted for most female rats (including controls) throughout the course of the study; a few male rats (including controls) exhibited this red-brown stain around eyes on days 2 and 21 of the study. No other clinical observations were noted.

Dermal irritation:

no effects observed

Description (incidence and severity):

There were no indications of skin irritation based on dermal scores.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

All groups of rats lost weight (2-4%) during the first week of the study. During the second week, however, all groups recovered the lost weight and continued to gain until study termination. Mean body weights were comparable between dose groups ,throughout the course of the study. The initial weight loss was probably the result of stress from the increased handling associated with the bandaging technique.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean feed consumption values were comparable between groups over the study period. Feed consumption was lower for all groups during the first week of the study but increased during the last 2 weeks of the study supporting the theory of initial stress associated with acclimation to the application technique.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects observed in routine hematology parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects observed in routine clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis values for chlorpyrifos treated groups of animals were comparable to controls.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Results of the functional observational battery indicate no appreciable difference between dose groups and control groups. Observations of decreased muscle tone noted for two female rats of the 5 mg/kg/day dose group and increased fecal staining for one male rat of the 0.5 mg/kg/day group were considered incidental findings since no treatment-related effects were observed in other parameters (specifically cholinesterase data).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no indications of systemic toxicity based on terminal body weight or organ weight data.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No indication of toxicity was observed during gross pathologic examination of animals treated dermally with chlorpyrifos for 21 days. Likewise, histopathologic examination did not reveal evidence of toxicity. Each of the few observations made at necropsy was present only in a single rat and all observations were considered to be spontaneous changes unrelated ·to treatment. In addition, all histopathologic observations (except for one control female rat) were of a minimal nature and were considered to be typical spontaneous changes normally present in rats of this strain and age. One female control rat had ureteral and urinary bladder calculi with associated hyperplasia and inflammation of the associated tissues and kidney.

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically identified differences in mean values of brain, plasma, or RBC cholinesterase activity for any group treated with chlorpyrifos. While values for 5.0 mg/kg/day male plasma and RBC cholinesterase and female plasma cholinesterase activities were 7, 11 and 17% lower, respectively, than their concurrent control groups, these values were within the range observed for recent historical controls in this laboratory.

Key result

Dose descriptor:

NOAEL

Effect level:

> 5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted after 21 day dermal exposure at highest level tested

Conclusions:

21-day dermal NOEL (female rat) > 5 mg/kg/day
However, application of 10 mg chlorpyrifos/kg body weight/day for 4 days, produced decreases in plasma (45%) and RBC (16%) cholinesterase activity without evidence of toxicity.

Executive summary:

In a probe study, female Fischer 344 rats {4/dose group) were exposed dermally to chlorpyrifos in corn oil solution at dose levels of 0 (corn oil), 1, 10, 100 or 500 mg/kg body weigh~/day, approximately 6 hours/day, for 4 consecutive days. Clinical observations were recorded daily, body weights were recorded on days 1 and 4, plasma and red blood cell (RBC) cholinesterase activities were measured and a gross necropsy conducted on day 5. There were substantial dose-related decreases relative to control in plasma cholinesterase activities (45, 91, or 98%) for animals that received 10, 100 or 500 mg/kg/day, respectively; RBC cholinesterase activities were also decreased in these groups (16, 49 or 75%, respectively). Animals that received 1 mg/kg/day were unaffected by treatment. Decreases in plasma cholinesterase activity as high as 98%

were not asociated with any changes in body weight, in-life clinical signs, or gross

pathologic alterations.

 

Based on the results of the probe study, 5 rats/sex/exposure group were subsequently exposed to a (corn oil), 0.1, 0.5, 1 or 5 mg chlorpyrifos/kg body weight/day in corn oil solution for approximately 6 hours/day, 5 days/week for 3 consecutive weeks. Body weight, feed consumption, in-life clinical observations, clinical laboratory studies, gross pathology and histopathology were evaluated and found to be unaffected. Plasma, RBC and brain cholinesterase activities measured at necropsy were comparable in treated and control groups. In addition, nothing remarkable was observed in a functional observational battery conducted immediately prior to necropsy.

 

Daily dermal application of up to 5 mg chlorpyrifos/kg body weight/day over 21 days (15

applications) was well tolerated by rats with no indication of systemic toxicity and no decrease in cholinesterase activity, the most sensitive indicator of exposure. However, application of 10 mg chlorpyrifos/kg body weight/day for 4 days, produced decreases in plasma (45%) and RBC (16%) cholinesterase activity without evidence of toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 mg/cm²

Study duration:

subacute

Species:

rat

Additional information

Repeated dose toxicity studies were conducted via the oral, dermal and inhalation routes of exposure. The results from the studies in rat, mouse and dog demonstrate that changes in AChE levels is the most sensitive toxicity end-point. 

 

In a 13-week rat toxicity study, Chlorpyrifos was fed at targeted dose levels of 0 (controls), 0.1, 1, 5, or 15 mg/kg/day. The primary effects of administration of 15 mg/kg/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/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/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/day level. 

 

In the 2-year dog study the NOAEL for RBC ChE inhibition was 0.1 mg/kg bw/day. Aside from cholinesterase inhibition no other toxicological effect was detected in any of the dogs receiving diets containing chlorpyrifos including clinical signs. No significant clinical toxicological effects were detected in any of the dogs receiving the diets containing chlorpyrifos. No test-material-related effects were seen on body weight, food consumption, haematological investigation, clinical chemistry or urinalysis. No test-material-related effects were seen on organ weights or histopathology in any part of the study.

 

In the majority of rat inhalation studies no effects were observed at the highest attainable chlorpyrifos concentration in air. One of them was conducted for 13 weeks where chlorpyrifos was exposed nose-only at 0, 5.2, 10.3 or 20.6 ppb (0, 75, 147 or 296µg/m3) for 6 hrs/day, 5 days/week for 13 weeks. The exposure concentrations were limited by the low vapor pressure of chlorpyrifos (theoretical maximum vapor concentration of 25 ppb at 25oC). No treatment-related signs of toxicity or changes in body weights were detected during the course of the study. Urinalysis, Hematology, clinical chemistry, organ weights, 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 chlorpyrifos was greater than the highest attainable concentration, 20.6 ppb in male and female rats.  

 

In a 21-day dermal toxicity study no treatment-related effects were observed at any dose and erythrocyte and brain acetylcholinesterase activities were not inhibited. In the 4-day range finding component of this study, decreased plasma cholinesterase activity (45%) and erythrocyte acetylcholinesterase activity (16%) were observed at 10 mg/kg bw/day, in the absence of clinical signs of intoxication.

Justification for classification or non-classification

Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health. There is no evidence for any adverse findings or serious target organ toxicity in 90-day repeat dosing studies in rats, mice or dogs that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. Apart from depression of body weights, depression of cholinesterase levels in the brain, plasma, and erythrocytes, and microscopically-detectable fatty vacuolization of the adrenal zona fascicula, no other serious effects were observed. So, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.