Chlorpyrifos

Neurotoxicity

Description of key information

Study Type	Species	Findings	Guideline	Reliability
13-week Oral Feed study	Rat	NOEL = 1 mg/kg	EPA FIFRA 82-7	1
Acute Oral Gavage	Rat	NOEL = 10 mg/kg bw	EPA FIFRA 82-7	1
Short-term oral Oral Gavage study	Rat	NOEL = 1 mg/kg	EPA FIFRA 82-7	1
Acute Oral Gavage	Rat, female	NOEL = 1 mg/kg	no guideline	2
2-week Oral Feed study	Rat	LOEL = 5 mg/kg bw/day	no guideline	2
13-week Oral Gavage	Hen	NOAEL = 10 mg/kg bw	no guideline	2
6-week Oral Feed study	Dog	NOAEL - Acute: 2 mg/kg bw/day NOEL - repeat dose: 1 mg/kg bw/day	no guideline	2
Acute Inhalation	Rat	NOEC - brain AChE: 22.1 mg/m³ air NOEC - RBC AChE: 3.7 mg/m³ air	no guideline	2
Acute Inhalation	Rat, female	NOEL: 0.254 mg/m³ air	no guideline	2
Acute Oral Gavage	Rat	NOEL: 0.5 mg/kg bw/day (nominal) based on: (test mat.) AChE inhibition: (With milk as a vehicle,no discernable gender differences in ChE activity in male or female PND 11 pups. Compared to corn oil, the maximum ChE inhibition at the time-of-peak effect for chlorpyrifos was slightly later in milk (8 h) than corn oil (6 h). - At the time-of-peak inhibition, the plasma chlorpyrifos and chlorpyrifosoxon levels, as well as the magnitude of ChE inhibition across tissues, were similar with both milk and corn oil vehicles.) NOEL: 0.1 mg/kg bw/day (nominal) based on: (test mat.)	no guideline	2
Short-term oral Oral Gavage study	Rat	ChE levels of all tissues of 5mg/kg bw/day pups rapidly returned to near control levels by PND5, further indicating a lack of differential sensitivity at this dosage level	no guideline	2

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA FIFRA Guideline No. 82-7

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot Number: MM-890115-616
Purity: 98.1 ± 0.3%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days a week

Dose / conc.:

0.1 mg/kg bw/day

Dose / conc.:

1 mg/kg bw/day

Dose / conc.:

5 mg/kg bw/day

Dose / conc.:

15 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A low incidence of perineal soiling was seen in female rats during clinical examinations (a 5 mg female at weeks 12 and 13; and a 15 mg female at week 13). This was considered to be related to treatment because it was consistent with similar observations made during the FOB. Other observations were not treatment related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on body weights at any time in either males or females.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

FOB hand-held and open field observations: Treatment-related perineal soiling was observed in a few female rats at 5 and 15 mg/kg.

Grip performance: There were no treatment-related effects on hindlimb or forelimb grip performance at any time during the study.

Landing foot splay: There were no treatment-related differences in landing foot splay.

Motor activity: Motor activity was decreased overall after sub-chronic treatment with 15 mg/kg/day of the test substance. The effects were mainly present during week 4 at the high dose, and were mitigated later on. There were no motor activity effects at lower dose levels at any time period. The test substance did not affect habituation of motor activity throughout epochs. The sexes had similar locomotor activity at all times in spite of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross lesions attributable to dosage with the test substance in either the perfused neuropathology group or the routine necropsy immersion fixed group.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no lesions observed in the central or peripheral nervous system which were attributable to treatment with the test substance at the highest dose tested, 15 mg/kg/day, in the diet for 13 weeks.

Key result

Dose descriptor:

NOEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Conclusions:

NOEL (rat): 1 mg/kg bw/day

Executive summary:

The study was conducted following EPA FIFRA guideline 82-7. Potential neurotoxicological effects of the test substance were evaluated in male and female Fischer 344 rats (10/sex/group) following a subchronic (13-week) dietary exposure, 7 days per week to 0, 0.1, 1, 5 or 15 mg/kg body weight/day. Parameters examined during the 13-week observation period included body weights, a functional observational battery (FOB) and motor activity. After completion of 13 weeks on study, histopathologic examinations of central and peripheral nervous tissues (perfusion fixed) were conducted.

The treatment-related findings included decreased motor activity (15 mg/kg high-dose group at week 4 only); and perineal soiling that occurred in a few female rats (5 mg group at weeks 8 and 13, and 15 mg group at weeks 4, 8 and 13). There were no other treatment-related findings in the FOB, and there were no treatment-related neuropathological alterations at any dose level.

It is known from other Fischer 344 rat dietary studies that oral test substance exposures of 5 and 15 mg/kg/day cause inhibition of brain and RBC acetylcholinesterase (AChE). RBC (but not brain) AChE also is inhibited at 1 mg/kg/day. In addition, adrenal morphology is altered at 5 and 15 mg/kg/day. Consequently, in the current study, the mild decrease in motor activity at 15 mg/kg/day likely was concurrent with significant reduction of brain and erythrocyte AChE; and with altered adrenal cortical morphology. Although the brain AChE likely was inhibited at 5 mg/kg/day, no motor activity effects occurred at this exposure level. The perineal soiling in a few female rats at 5 and 15 mg/kg/day was attributed to treatment, since clear inhibition of AChE occurs at these exposure levels. The no-observed-effect level (NOEL), consequently, was 1 mg/kg/day.

Reference 2

Endpoint:

neurotoxicity: short-term 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:

other: EPA FIFRA Guideline No. 82-7

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Substance ID: TSN 100227
Lot Number: MM930503-17
Purity: 99.8 ± 0.1%

Species:

rat

Strain:

other: Crl:CD®BR VAF/Plus® (Sprague-Dawley)

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Gestation day 6 through lactation day 11

Frequency of treatment:

Once daily

Dose / conc.:

0.3 mg/kg bw/day

Dose / conc.:

1 mg/kg bw/day

Dose / conc.:

5 mg/kg bw/day

No. of animals per sex per dose:

25 (main test)
5 (satellite group)

Control animals:

yes, concurrent vehicle

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Dams at highest dose (5 mg/kg bw/day): Small decreases in body weight gain and a reduction (4.2 to 14.5%) in feed consumption during the lactation dosing. Maternal toxicity (muscle fasciculations, hyperpnea, hyperreactivity) mostly during the lactation period, but fasciculations also occurred late in the gestation period. Brain cholinesterase inhibited by ~90% (pup brain cholinesterase was not measured in this study).

Pups at highest dose (5 mg/kg bw/day): More litters with pups dying between DPs 1 and 5 (decreased viability index). Lower body weight (~5 to 20%) over all time intervals (between DPs 1 and 66) and decreased postweaning food consumption; delayed pinna detachment, vaginal patency, preputial separation; in the absence of neuropathological findings, a transient decrease in brain weight and layer thickness (cerebellum in males; parietal cortex, caudateputamen, hippocampal gyrus in females), with a concomitant increase in brain-to-body-weight ratio (present on DP 12, but not on DPs 66 to 71); increased latency of the auditory startle response (DP 23 but not on DP 62).

No effects were seen in the pups in the absence of maternal toxicity. In the high dosage group, the lower birth weights and delayed developmental landmarks were indicative of a maturational delay, also reflected in the decreased brain weights and linear dimensions of some neuroanatomic areas on DP 12. Cognitive functions (i.e., learning, memory, habituation) were not affected by the test substance. The changes in the auditory startle data were present on DP 23, but had receded by DP 60, and were attributed to motor slowing.

Details on results:

In summary, the test substance did not cause any effects in the offspring when administered to dams during gestation and lactation at dose levels of 0.3 or 1 mg/kg/day. Administration of the test substance at a dose level of 5 mg/kg/day produced evidence of cholinergic toxicity in the dams. Increased pup mortality, transient decrease in pup body weight, brain size, brain layer thickness, transient changes in the startle response, and delayed pinna detachment, vaginal opening and preputial separation were seen in the presence of maternal toxicity and were consistent with delayed maturation. Cognitive functions (learning, memory, habituation) were not impaired in the pups at any time at any dosage. These data demonstrate that the test substance was not a selective developmental neurotoxicant in the rat.

Key result

Dose descriptor:

NOEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

other: for maternal and pup toxicity

Conclusions:

NOEL (rat): 1 mg/kg/day

Executive summary:

The study was conducted following following EPA FIFRA guideline 82-7. The purpose of this study was to evaluate the potential for the test substance to cause developmental neurotoxicity in offspring when administered by gavage from gestation day 6 through lactation day 11 (birth being lactation day 1) to rat dams at dosages of 0, 0.3, 1 and 5 mg/kg/day. The test substance did not cause any effects in the offspring when administered to dams during gestation and lactation at dosage levels of 0.3 or 1 mg/kg/day. No effects were detected in pups at dosages that were not maternally toxic. A developmental delay and pup decreased viability were observed at a maternally toxic dosage (i.e., 5 mg/kg/day) and were consistent with undernutrition and potentially altered maternal care of the pups. Lower body weight gains were accompanied by decreased food consumption and by a transient decrease in brain weight and layer thickness of some brain areas in either male or female pups (in the absence of neuropathology). Increased auditory startle latency and, possibly, decreased peak response were documented in the pups, consistent with previously reported motor slowing. Cognitive functions (learning, memory, habituation) were not impaired in the pups at any time at any dosage. These data demonstrate that the test substance was not a selective developmental neurotoxicant in the rat. The no-observed-effect-Ievel (NOEL) for maternal and pup toxicity in this study was 1 mg/kg/day.

Reference 3

Endpoint:

neurotoxicity: acute 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:

other: EPA FIFRA Guideline No. 82-7

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot Number: MM-890115-616
Purity: 98.1 ± 0.2%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Dose / conc.:

10 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

100 mg/kg bw (total dose)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related increase in perineal soiling was observed in male and female rats on day 2. Incidences of perineal soiling also were seen on day 3. Low incidences of decreased activity were observed in treated males and females on day 2. One male rat of the high dose group had excessive chromodacryorrhea on day 2; another rat in this group showed chromorhinorrhea. Excessive chromodacryorrhea and chromorhinorrhea also were seen in treated females on days 2, 3 and 4. One female rat of the high dose group had decreased muscle tone on days 2 and 3. Some female rats of the high dose group were hyperreactive to stimuli on days 2 to 5. All rats appeared clinically normal from day 6 to the end of the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment had an overall statistically significant (p=0.0001) effect on body weight. An overall treatment-by-day-by-sex interaction (p= 0.0006) also was observed. ANOVA analysis for each test day showed a statistically significant effect on body weights for test day 2 only (p= 0.0001 for mid- and high dose groups). No treatment-related differences in body weight were observed during the prestudy period or on test days 1,8 or 15.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Handheld and open field observations: During the FOB on day 1 (approximately 6 hours after dosing), treatment-related effects were seen in handheld and open field observations. No FOB effects were noted on days 8 or 15. Day 1 FOB changes in high dose females included: incoordination, decreased muscle tone, tremor, increased lacrimation and salivation, decreased urination and defecation, and slightly more perineal soiling compared to the controls. Male rats were less affected by treatment than females; a few high dose males had increased lacrimation, reduced reactivity to handling, one had tremor, one was incoordinated, and a couple had equivocally reduced response to tail pinch and reduced defecation.

Grip performance: For females, visual examination of the day 1 pattern across doses suggests a mild effect at the middle and high doses. For males, the day 1 dose-pattern lacks dose-response, and the relationship of low-dose performance to treatment is unclear. Day 1 effects were non-significant in males (p = 0.1469), and, were statistically suggestive in females (p = 0.0471). Analysis of female day 1 data by dose level suggested a treatment effect only at the high dose (p = 0.0257).

Landing foot splay: On day 1 (day of dosing), hindlimb landing foot splay was severely affected by treatment in the high dose females. 6 out of 10 females of the high dose group were not able to perform the landing foot splay procedure.
No significant changes in any of the FOB or clinical parameters were observed on day 15 of the study period.

Motor activity: A statistically significant decrease in motor activity was observed at 50 and 100 mg/kg on day 1, approximately 5 to 6 hours after dosing. On day 8, motor activity was significantly reduced at 50 and 100 mg/kg in females only. No statistically significant changes in motor activity were observed on day 15 of the study.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The results of this histologic evaluation do not indicate that the test substance had any effect on the central and peripheral nervous system.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Conclusions:

NOEL (rat): 10 mg/kg

Executive summary:

Neurotoxicological effects of the test substance were evaluated following EPA FIFRA guideline 82-7 in male and female Fischer 344 rats following a single-dose exposure by oral gavage to 0, 10, 50 or 100 mg/kg. Parameters examined during the two-week observation period included body weights, a Functional Observational Battery (FOB), motor activity and neuropathology. The FOB consisted of handheld and open field observations, grip performance and landing foot splay testing. Ten animals/sex/group were evaluated by FOB and motor activity assay; once prior to exposure, on day 1 at the time of peak effect (approximately 6 hours after dosing), and on days 8 and 15 of the study period. Body weights were determined on days -1, 1, 2, 8, and 15. After completion of 2 weeks of study, neuropathology of central and peripheral nervous tissues was conducted on 5 animals/sex/group.

Prominent FOB changes occurred in high-dose rats on day 1 only, females were more affected than males. Only females had FOB changes at the middle dose. Female mid- and high dose rats had a mild decrease in grip performance, and high-dose females had a severe effect on hindlimb splay. No FOB effects were noted at the low dose. The day 1 effects were consistent with those expected of acute organophosphate overexposure. It should be noted that, on day 1, the FOB was conducted at the time of peak effects as determined by preliminary dose range-finding study. Hand-held clinical examinations on days 2, 3 and 4 indicated diminishing clinical signs of treatment to near normal by day 4. The FOB conducted on day 8 indicated the rats at all dose levels appeared clinically normal by this time. Body weights of both high- and mid-dose males and females were significantly reduced on day 2 of the test period. Body weights were not significantly altered on days 8 or 15. No significant changes in any of the FOB or clinical parameters were observed on day 15 of the study period. A statistically significant decrease in motor activity was observed at 50 and 100 mg/kg on day 1, approximately 5 to 6 hours after dosing. On day 8, motor activity was significantly reduced at 100 mg/kg in females only. No statistically significant changes in motor activity were observed on day 15 of the study. Histologic evaluation of the central and peripheral nervous systems did not reveal any treatment-related effects.

The results of this study indicated a No-Observed Effect Level (NOEL) of 10 mg/kg.

Reference 4

Endpoint:

neurotoxicity: sub-chronic 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:

Ch1orpyrifos was evaluated to identify any propensity to cause organophate-induced de1ayed-neurotoxicity by subchronic administration. Chlorpyrifos was given in corn oil via gavage to laying chicken hens (10/group) at dose levels of 0 (negative control), 0 (positive control),
1, 5, or 10 mg chlorpyrifos/kg body weight/day, seven days/week, for 13 weeks.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

DURSBAN F
AGR-214637, Lot #MM820905-610
Purity: 95.7%

Species:

hen

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7/days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

The birds were observed at least once daily. Body weights were recorded weekly. As part of the assessment of general health and laying status (not as a measure of delayed-neurotoxicity), the number of eggs found daily in each pen was recorded.

For the purpose of detecting sUbtle signs suggestive of clinical delayed-neurotoxicity, the birds of all groups were.observed weekly while being induced to walk about on a bare concrete floor and to climb a short, inclined (30°) ramp. These observations were made starting the third week of the study.

Sacrifice and (histo)pathology:

All birds survived until scheduled necropsy on Study Days 92, 93 and 95, at which time each was anesthetized with an intramuscular injection of ketamine hydrochloride followed by methoxyflurane. An incision was then made in the abdominal musculature, and the heart was visualized. A blunted needle was placed in the left ventricle, an incision made in the right atrium, and the bird was perfused with a heparinized saline solution followed by 200-300 ml neutral phosphate-buffered 10% formalin. The cranium and spinal column were removed in toto and placed in a jar containing formalin. Both sciatic nerves and both tibial nerves were dissected free from the musculature, secured on wooden tongue depressors , and immersed in formalin.

For histology, sections taken included the following: one cross-section of brain through the cerebellum and anterior medulla; a cross-section
through the posterior medulla at the obex; a cross-section and longitudinal section of the cervical spinal cord between C3 and C6, the
thoracic spinal cord at the brachial bulb, and the lumbosacral spinal cord at the lumbosacral bulb anterior to the glycogen body; a crosssection
and longitudinal section of both sciatic nerves; and an approximately 1 cm longitudinal section of both tibial nerves. Tissues were processed conventionally, sectioned at 5-6 microns, stained with hematoxylin and eosin, luxol fast blue-periodic acid-Schiff for myelin, and Sevier-Munger silver stain for axons, and examined microscopically. Only tissues from birds administered the positive control (10 mg TOCP/kgBW/day), the negative control (corn oil only), and the high dose level test material (10 mg ch1orpyrifos/kgBW/day) were examined histologically. All sections were examined blind to treatment and scored, according to a classification scheme modified from Bickford and Sprague and Prentice and Roberts..

Positive control:

tri-ortho-cresy1-phosphate (TOCP)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The birds of the high dose chlorpyrifos group (10 mg/kgBW/day) lost weight. relative to control. starting in the first week of the study. Some of the birds of this group developed transient (lasting fewer than 24 hours) signs indicative of acute organophosphate toxicity. However, none of the birds treated with chlorpyrifos including the birds of the high dose level group. showed any clinical signs or histopathological lesions characteristic of OPION.

Body weight and weight changes:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no neurotox effects at highest dose tested

Conclusions:

The test substance did not induce delayed neurotoxicity when given orally at dose levels up to 10 mg/kgBW/day, seven days/week, for 13 weeks.

Executive summary:

Ch1orpyrifos was evaluated to identify any propensity to cause organophate-induced de1ayed-neurotoxicity by subchronic administration. Chlorpyrifos was given in corn oil via gavage to laying chicken hens (10/group) at dose levels of 0 (negative control), 0 (positive control), 1, 5, or 10 mg chlorpyrifos/kg body weight/day, seven days/week, for 13 weeks.

 

Under the same regimen. tri-ortho-cresy1-phosphate (TOCP; 10 mg/kgBW/day) was used as the positive control; the negative control group received corn oil only. No protection (e.g., atropine) was given against possible acute cholinergic response. The primary criterion for OPIDN was defined as the microscopic detection of characteristic lesions and, to this end, central and peripheral nervous system tissue from all birds of the negative control, positive control, and the high dose chlorpyrifos (10 mg/kgBW/day) groups was examined mjcroscopically. Other, secondary, variables were general health status, body weights. and clinical signs associated with OPION. The birds of the high dose chlorpyrifos group (10 mg/kgBW/day) lost weight. relative to control. starting in the first week of the study. Some of the birds of this group developed transient (lasting fewer than 24 hours) signs indicative of acute organophosphate toxicity. However. none of the birds treated with ch1orpyrifos including the birds of the

high dose level group. showed any clinical signs or histopathological lesions characteristic of OPION.

 

After about 5 weeks of treatment. birds of the positive control group (10 mg.TOCP/kgBW/day) started to lose weight and to develop the clinical signs of OPION; poststudy evaluation showed the central and peripheral nervous system lesions characteristic of OPION. It was concluded that. under the conditions of this study. Chlorpyrifos insecticide did not induce subchronic OPION when given orally at dose levels up to 10 mg/kgBW/day, seven days/week, for 13 weeks.

Reference 5

Endpoint:

neurotoxicity: acute 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:

The purpose was to evaluate the dose response for inhibition of the enzymes cholinesterase (ChE) and neurotoxic esterase (NTE) in female Fischer 344 rats receiving single oral doses of the organophosphorus compound chlorpyrifos. Six rats/dose level were administered 0, 0.5, I, 5, 10, 50 or 100 mg/kg of chlorpyrifos by single oral gavage following fasting overnight. Assays of brain NTE and plasma, RBC, heart muscle and brain ChE were conducted 24 hours post-gavage.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Technical grade Chlorpyrifos (DURSBANI F insecticide)
Lot # MM-890115-616
Purity: 98.1%

Species:

rat

Strain:

Fischer 344

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Once

Frequency of treatment:

Dosing was staggered over 2 days to accommodate running of the enzyme assays

Dose / conc.:

0 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)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

Animals were observed cageside twice daily.

Body weights were taken prior to randomization and once prior to dosing.

Specific biochemical examinations:

Assays of brain NTE and plasma, RBC, heart muscle and brain ChE were conducted 24 hours post-gavage.

Clinical signs:

no effects observed

Body weight and weight changes:

not examined

Description (incidence and severity):

Body weight data were not statistically analyzed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Neurotoxic esterase activity of brain homogenates showed no statistically significant differences from the control group at any dose level. Treatment-related inhibition of plasma, red blood cell, and heart cholinesterase was observed in rats dosed with 5, 10, 50 or 100 mg/kg of chlorpyrifos. Brain cholinesterase showed a statistically significant inhibition only at dose levels of 50 or 100 mg/kg of
chlorpyrifos.

Key result

Dose descriptor:

NOEL

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

Conclusions:

Acute neurotoxic NOEL (female Fischer 344 rats) = 1 mg/kg based on plasma, red blood cell and heart ChE inhibition at 5 mg/kg.

Executive summary:

The purpose was to evaluate the dose response for inhibition of the enzymes cholinesterase (ChE) and neurotoxic esterase (NTE) in female Fischer 344 rats receiving single oral doses of the organophosphorus compound chlorpyrifos. Six rats/dose level were administered 0, 0.5, 1, 5, 10, 50 or 100 mg/kg of chlorpyrifos by single oral gavage following fasting overnight. Assays of brain NTE and plasma, RBC, heart muscle and brain ChE were conducted 24 hours post-gavage.

 

Neurotoxic esterase activity of brain homogenates showed no statistically significant differences from the control group at any dose level. Treatment-related inhibition of plasma, red blood cell, and heart cholinesterase was observed in rats dosed with 5, 10, 50 or 100 mg/kg of chlorpyrifos. Brain cholinesterase showed a statistically significant inhibition only at dose levels of 50 or 100 mg/kg of

chlorpyrifos. The no-observed-effect level (NOEL) was 1 mg/kg based on plasma, red blood cell and heart ChE inhibition at 5 mg/kg.

Reference 6

Endpoint:

neurotoxicity: acute 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:

In the acute comparative cholinesterase study, initial studies were conducted to determine the time-of-peak inhibition for red blood cell (RBC), brain and plasma cholinesterase (ChE) in pups and adults. Groups of 4-6 PND 11 female CD rat pups were administered a single gavage dose (corn oil vehicle) of 0 or 3 mg/kg chlorpyrifos or 0 or 0.5 mg/kg chlorpyrifos-oxon. A separate group of PND 11 female pups (6/dose) were exposed to 0 or 5 mg/kg chlorpyrifos in rat milk to compare the impact of vehicle on ChE inhibition. Similarly, adult female CD rats (4/group) were administered a single gavage dose (cornvoil vehicle) of 0 or 10 mg/kg chlorpyrifos or 0 or 0.3 mg/kg chlorpyrifos-oxon. A separate group of adult females were exposed for 12-h to dietary chlorpyrifos (dose levels of 0 or 10 mg/kg) to compare acute ChE inhibition by a more relevant exposure scenario.

In the repeated dose studies, ChE activity was measured in PND 21 male and female CD rat pups (8/sex/dose) and adult (~PND 80) female CD rats (8/dose) following exposure to eleven daily gavage doses (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 1, 3.5 mg/kg/day chlorpyrifos or 0.01 or 0.5 mg/kg/day chlorpyrifos-oxon.

GLP compliance:

yes

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419
Purity: 99.8%

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Route of administration:

other: gavage and feeding

Vehicle:

other: corn oil or rat milk

Remarks:

for the gavage portion of the study

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

single gavage dose or 12-hour feeding

Frequency of treatment:

once

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (corn oil vehicle) PND 11 female pups

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (corn oil vehicle) PND 11 female pups

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (rat milk vehicle) PND 11 female pups

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (rat milk vehicle) PND 11 female pups

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (adult females)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Prelim study: gavage (adult females)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Prelim study: feeding (adult females)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Prelim study: feeding (adult females)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Definitive acute study

Dose / conc.:

0.05 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (male/female pups)

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (male/female pups)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (male/female pups)

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (male/female pups)

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (male/female pups)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Definitive gavage (female adult)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Repeat dose study

Dose / conc.:

0.05 mg/kg bw/day (nominal)

Remarks:

repeat dose study

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Remarks:

repeat dose study

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

repeat dose study

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

repeat dose study

Dose / conc.:

3.5 mg/kg bw/day (nominal)

Remarks:

repeat dose study

No. of animals per sex per dose:

Prelim studies:
PND11 pups (corn oil) = 4-6/group
PND 11 pups (milk) = 6/group
adult (gavage) = 4/group
adult (feeding) = 4/group

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

Clinical observations were recorded pre- and post-dosing; body weights and feed consumption were monitored for adults exposed to dietary chlorpyrifos.

Specific biochemical examinations:

In both pups and adults, samples (RBC, brain, plasma) were collected at 2, 4, 6, 8, 12 , 24 or 48 h postdosing and ChE inhibition was determined using a modified Ellman method.

Neurobehavioural examinations performed and frequency:

In the repeat dose study, Animals were given daily clinical observations and body weights during the exposure period. Neurotoxicity was assessed at the time-of-peak ChE inhibition after the 10th exposure by performing a functional observational battery and examining motor activity in PND 20 pups and ~PND 79 adults.

Key result

Dose descriptor:

NOEL

Effect level:

0.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: AChE inhibition across all tissues in both age groups with acute exposure

Key result

Dose descriptor:

other: AChE inhibition

Remarks on result:

other: With milk as a vehicle,no discernable gender differences in ChE activity in male or female PND 11 pups. Compared to corn oil, the maximum ChE inhibition at the time-of-peak effect for chlorpyrifos was slightly later in milk (8 h) than corn oil (6 h).

Remarks:

At the time-of-peak inhibition, the plasma chlorpyrifos and chlorpyrifosoxon levels, as well as the magnitude of ChE inhibition across tissues, were similar with both milk and corn oil vehicles.

Key result

Dose descriptor:

NOEL

Effect level:

0.1 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: ChE inhibition across all tissues in both age groups with repeated exposure

Conclusions:

Young pups (PND 11, and PND 11-21) are not more sensitive to ChE inhibition over the lower portion of the dose-response curves. These data suggest that infants and children would not be more sensitive to chlorpyrifos-induced ChE inhibition at environmentally relevant doses.

Executive summary:

In the acute comparative cholinesterase study, initial studies were conducted to determine the time-of-peak inhibition for red blood cell (RBC), brain and plasma cholinesterase (ChE) in pups and adults. Groups of 4-6 PND 11 female CD rat pups were administered a single gavage dose (corn oil vehicle) of 0 or 3 mg/kg chlorpyrifos or 0 or 0.5 mg/kg chlorpyrifos-oxon. A separate group of PND 11 female pups (6/dose) were exposed to 0 or 5 mg/kg chlorpyrifos in rat milk to compare the impact of vehicle on ChE inhibition. Similarly, adult female CD rats (4/group) were administered a single gavage dose (corn oil vehicle) of 0 or 10 mg/kg chlorpyrifos or 0 or 0.3 mg/kg chlorpyrifos-oxon. A separate group of adult females were exposed for 12-h to dietary chlorpyrifos (dose levels of 0 or 10 mg/kg) to compare acute ChE inhibition by a more relevant exposure scenario.

 

Clinical observations were recorded pre- and post-dosing; body weights and feed consumption were monitored for adults exposed to dietary chlorpyrifos. In both pups and adults, samples (RBC, brain, plasma) were collected at 2, 4, 6, 8, 12 , 24 or 48 h postdosing (48 h included for PND 11 pups given CPF in corn oil and CPF in milk only) and ChE inhibition was determined using a modified Ellman method.

 

There were no treatment-related clinical observations in either pups or adults using these dosing scenarios. Using experimental data, published literature and physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model, time–to-peak ChE inhibition was determined for each dosing scenario. For PND 11 pups, the times-to-peak inhibition were 6 h post-dosing for chlorpyrifos in corn oil, 4 h post-dosing for chlorpyrifos-oxon in corn oil and 8 h post-dosing for chlorpyrifos in milk. For adult female rats, the times-to-peak inhibition were 8 h post-dosing for chlorpyrifos in corn oil, 4 h post-dosing for chlorpyrifos-oxon in corn oil, and 8 h post-exposure for chlorpyrifos in diet (i.e., after conclusion of the 12-h dietary exposure to chlorpyrifos-containing diet).

 

The definitive acute comparative cholinesterase study examined age-related differential sensitivity across multiple dose levels of chlorpyrifos. ChE activity was measured in PND 11 male and female CD rat pups (8/sex/dose) and adult female CD rats (8/dose) exposed to a single gavage dose (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 2, 5 (pups only) or 10 (adults only) mg/kg chlorpyrifos. There were no discernable gender differences in ChE activity in male or female PND 11 pups in response to chlorpyrifos treatment. Both pups and adults had significant and comparable decreases in brain ChE activity at the highest dose of chlorpyrifos (5 mg/kg in PND 11 pups and 10 mg/kg in adults) and significant RBC and plasma ChE inhibition at doses >2 mg/kg (Summary Table 1). The 0.5 mg/kg chlorpyrifos dose was considered a no-observed-effect-level (NOEL) for ChE inhibition across all tissues in both age groups with acute exposure. Thus, pups may be more sensitive to ChE inhibition at higher dose levels of chlorpyrifos (e.g., 5 mg/kg) due to higher plasma levels of chlorpyrifos and its metabolites; however, pups are equally sensitive or less sensitive than adults at lower chlorpyrifos levels (e.g., 0.5 mg/kg). Thus, there was no evidence of age-related differential sensitivity to acute chlorpyrifos exposure over the lower portion of the dose-response curve.

For the definitive acute comparative cholinesterase study with chlorpyrifos-oxon, ChE activity was measured in PND 11 male and female CD rat pups (8/sex/dose) and adult female CD rats (8/dose) exposed to a single gavage dose (corn oil vehicle) of 0, 0.005 (pups only), 0.01, 0.05, 0.1, 0.5, or 1 (adults only) mg/kg chlorpyrifos-oxon. There were no discernable gender differences in ChE activity in male or female PND 11 pups. There was no significant inhibition of brain ChE in either pups or adults at any dose level of chlorpyrifos-oxon (Summary Table 2). RBC ChE inhibition was seen at the same dose level (0.5 mg/kg) in PND 11 pups and adults, but PND 11 pups exhibited significant be related to the 1.6X higher plasma TCP levels in pups at this dose level (both adult and pup samples collected at 4 h post-exposure; chlorpyrifos-oxon was below the lower limit of quantitation in pups and adults). Thus, the 0.1 mg/kg chlorpyrifos-oxon dose was considered a NOEL for ChE inhibition in RBC and brain in PND 11 pups and a NOEL in all tissues for adult females with acute exposure.

To examine a more relevant dosing scenario for pups, ChE activity was measured inPND 11 male and female CD rat pups (8/sex/dose) exposed to a single gavage dose (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 2, or 5 mg/kg chlorpyrifos. When using milk as a vehicle, there were no discernable gender differences in ChE activity in male or female PND 11 pups in response to chlorpyrifos treatment. Compared to corn oil, the maximum ChE inhibition at the time-of-peak effect for chlorpyrifos was slightly later in milk (8 h) than corn oil (6 h). At the time-of-peak inhibition, the plasma chlorpyrifos and chlorpyrifosoxon levels, as well as the magnitude of ChE inhibition across tissues, were similar with both milk and corn oil vehicles.

 

To examine a more relevant dosing scenario for adults, females (~ 70 days of age, 8/dose) were exposed for 12-h to dietary chlorpyrifos at doses of 0, 0.05, 0.1, 0.5, 2 or 10 mg/kg and acute ChE inhibition was compared to levels seen in adult females exposed to the same chlorpyrifos doses by gavage in corn oil. With a 12 h dietary exposure to chlorpyrifos, adult females exhibited significant brain ChE inhibition at the same concentration as chlorpyrifos given via gavage in corn oil (10 mg/kg); however, the magnitude of brain ChE inhibition was less by the dietary route, where plasma chlorpyrifos and TCP levels were 7.4X and 2.4X lower, respectively. This result suggests that dose rate impacts plasma levels of chlorpyrifos and its metabolites and subsequently, the magnitude of brain ChE inhibition. In contrast RBC ChE inhibition appeared to be more pronounced at 2 mg/kg using the dietary exposure scenario. In both dosing scenarios, ChE inhibition was not significantly altered in any tissue at the 0.5 mg/kg dose.

 

In the repeated dose studies, ChE activity was measured in PND 21 male and female CD rat pups (8/sex/dose) and adult (~PND 80) female CD rats (8/dose) following exposure to eleven daily gavage doses (corn oil vehicle) of 0, 0.05, 0.1, 0.5, 1, 3.5 mg/kg/day chlorpyrifos or 0.01 or 0.5 mg/kg/day chlorpyrifos-oxon. Animals were given daily clinical observations and body weights during the exposure period. Neurotoxicity was assessed at the time-of-peak ChE inhibition after the 10thexposure by performing a functional observational battery and examining motor activity in PND 20 pups and ~PND 79 adults.

 

Eleven-day exposures with chlorpyrifos or chlorpyrifos-oxon did not significantly affect clinical signs or neurobehavioral toxicity measures in either pups or adults, including no treatment-related effects on FOBs and motor activity assessments. There were no significant effects on body weights in pups or adults at any dose of chlorpyrifos or chlorpyrifos-oxon during the treatment periods. There were no discernable gender differences in ChE activity in male or female PND 11 pups in response to repeated chlorpyrifos treatment. At the high dose (3.5 mg/kg/day), pups had similar ChE inhibition to adults across all tissues, despite having higher plasma levels of CPF (Summary Table 3). The higher plasma levels in pups were likely related to a later sampling time in adults (8 h) vs. pups (6 h) relative to the proposed Cmaxfor gavage chlorpyrifos in corn oil (2-3 h) and a somewhat higher metabolic rate in adults. At 1 mg/kg/day, ChE was significantly decreased in both pups and adults across all tissues. At 0.5 mg/kg/day, there was no significant brain ChE inhibition in either adults or pups.

Both age groups had significant decreases in RBC and plasma ChE activity with no apparent difference in sensitivity. Thus, for a given plasma chlorpyrifos level, pups were equally sensitive or less sensitive than adults for ChE inhibition. As chlorpyrifos doses decreased below 1 mg/kg/day, there was no evidence of increased sensitivity in pups to chlorpyrifos-induced ChE inhibition. The 0.1 mg/kg/day chlorpyrifos dose was considered a no-observed-effect-level (NOEL) for ChE inhibition across all tissues in both age groups with repeated exposure.

The response of pups and adults to chlorpyrifos-oxon was similar with significant decreases in RBC and plasma ChE at 0.5 mg/kg/day in both age groups, although adults had 2X the levels of TCP as pups (Summary Table 4). ChE was not inhibited in either of these tissues at 0.01 mg/kg/day in either adults or pups. Brain ChE was not altered at either dose of chlorpyrifos-oxon; thus, the 0.01 mg/kg/day chlorpyrifos-oxon dose was considered a no-observed-effect-level (NOEL) for ChE inhibition across all tissues in both age groups with repeated exposure.

Overall, both the acute and repeated-dose data collected in this study support the conclusion that young pups (PND 11, and PND 11-21) are not more sensitive to ChE inhibition over the lower portion of the dose-response curves. These data suggest that infants and children would not be more sensitive to chlorpyrifos-induced ChE inhibition at environmentally relevant doses.

Reference 7

Endpoint:

neurotoxicity: short-term 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:

Chlorpyrifos was tested to establish a dose-response for anticholinesterase effects and to determine appropriate dose levels for a two-generation dietary reproduction study in Sprague-Dawley rats. Groups of 7 rats/ sex/ dose level were given diets that provided targeted dose levels of 0, 5, or 10 mg chIorpyrifos/kg body weight/ day for 2 weeks.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos (technical grade DURSBAN F insecticide
AGR 273801
Purity: 985%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

continuous

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, plain diet

Observations and clinical examinations performed and frequency:

All animals were observed daily for evidence of treatment-related alterations in behavior or demeanor. Body weights and feed consumption were
recorded. at 3-4 day intervals on all animals.

Specific biochemical examinations:

While the animals were anesthetized, a sample of blood was collected via orbital sinus puncture for plasma and RBC cholinesterase determinations. Subsequently, the anesthetized animals were sacrificed by decapitation. The brain and adrenals were removed and weighed, and one half of the brain was used for cholinesterase measurements. Brain, RBC and plasma cholinesterase concentrations were determined. using a photometric technique. Brain samples were prepared for cholinesterase determinations.

Sacrifice and (histo)pathology:

A complete necropsy was conducted on all rats. Following the dosing period, animals were fasted over night, weighed and anesthetized with methoxyflurane.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dose-related decreases in plasma, red blood cell (RBC) and brain cholinesterase were observed in males and females exposed to chlorpyrifos. At 10 mg/kg/ day, RBC and plasma cholinesterase were decreased approximately 60% to 75%, and brain cholinesterase levels were decreased approximately 65% in both males and females. At 5 mg/kg/day RBC and plasma cholinesterase levels in males and females were also depressed approximately 60% to 75%, whereas brain cholinesterase levels were depressed approximately 42% in males and females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative adrenal weights were significantly elevated among females, but not in males, given 10 mg/kg/ day.

Key result

Dose descriptor:

LOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Conclusions:

2-week dietary LOEL = 5 mg/kg/day based on decreases in brain, RBC and plasma cholinesterase levels

Executive summary:

Chlorpyrifos was tested to establish a dose-response for anticholinesterase effects and to determine appropriate dose levels for a two-generation dietary reproduction study in Sprague-Dawley rats. Groups of 7 rats/ sex/ dose level were given diets that provided targeted dose levels of 0, 5, or 10 mg chIorpyrifos/kg body weight/ day for 2 weeks.

 

Dose-related decreases in plasma, red blood cell (RBC) and brain cholinesterase were observed in males and females exposed to chlorpyrifos. At 10 mg/kg/ day, RBC and plasma cholinesterase were decreased approximately 60% to 75%, and brain cholinesterase levels were decreased approximately 65% in both males and females. At 5 mg/kg/day RBC and plasma cholinesterase levels in males and females were also depressed approximately 60% to 75%, whereas brain cholinesterase levels were depressed approximately 42% in males and females. Absolute and relative adrenal weights were significantly elevated among females, but not in males, given 10 mg/kg/ day. No other treatment-related effects were noted among males or females at any dose level.

Reference 8

Endpoint:

neurotoxicity: sub-chronic 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:

The present study measured AChE activity in peripheral tissues as well as in erythrocytes and the brain, as requested by the UK Advisory Committee on Pesticides. AChE activity was measured in peripheral tissues (nodose ganglia, left atrium, diaphragm, and quadriceps), red blood cells (RBC) and brain in male and female Beagle dogs (six months old, n = four/sex/group) exposed for six weeks to 0.0, 0.5, 1.0, or 2.0 mg/kg/day chlorpyrifos in their diets.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Dursban FM (Chlorpyrifos Technical)
Lot #: 7299412, TS100759)
Purity: 97.6%

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

7 days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4/sex/group

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

Clinical examinations were conducted on all animals pre-exposure and weekly throughout the study period.

All dogs were weighed during the pre-exposure period and twice weekly throughout the study.

Feed consumption data were collected pre-exposure and daily throughout the study.

Specific biochemical examinations:

AChE activity was measured in peripheral tissues (nodose ganglia, left atrium, diaphragm, and quadriceps), red blood cells (RBC) and brain in male and female Beagle dogs.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cage-side observations, conducted on all animals throughout the study, were handrecorded and held in the study file. These observations revealed multiple occurrences of soft feces in all groups during various days of the study and were therefore not considered a treatment-related effect. Examinations performed on all animals pre-exposure and throughout the study revealed no treatment-related effects.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weights of treated males were reduced during the first week of treatment. There was no association with dosage and differences from pre-exposure (day 1) values were too small « 3%) to be of any toxicological importance. Thereafter, mean body weight gains were comparable in control and all test groups. In females, inter-group differences in body weight showed no association with dosage, with the largest body weight gains occurring in high-dose animals.

Food consumption and compound intake (if feeding study):

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

RBC AChE activity showed a slight (ca. 10%) but statistically significant decrease in the mid- and high-dose groups following the first day's exposure. RBC AChE activity reached significantly decreased (> 20% inhibition) steady-state levels in all groups within three weeks of treatment.

The small difference in brain AChE activity between high-dose dogs (male and female combined) and controls (approximately 7% inhibition) may represent a true effect, although it was not statistically significant and was not adverse « 20%). Differences in brain AChE activity between mid-dose males and controls were statistically significant (p < 0.05) but were not considered an effect of treatment in light of the lack of effect at this dosage in all previous dog studies and in females in this study. The NOEL for brain AChE was 1.0 mg/kg/day.

AChE activity in diaphragm, quadriceps, and nodose ganglia showed no significant differences between treated and control groups for males or females. However, it is possible that the lower activity in the left atrium AChE activity in high-dose male dogs was due to treatment, despite the lack of statistical significance and a comparable but opposite effect in female dogs. Therefore, the NOAEL for Beagle dog left atrium AChE was at least 1.0 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Acute

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: RBC AChE

Key result

Dose descriptor:

NOEL

Remarks:

repeat dose

Effect level:

1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: brain and peripheral tissues (AChE)

Conclusions:

Acute NOAEL (dog) = 2.0 mg/kg for RBC AChE
Repeat dose NOEL (dog) = 1.0 mg/kg for brain and peripheral tissue AChE

Executive summary:

The present study measured AChE activity in peripheral tissues as well as in erythrocytesand the brain, as requested by the UK Advisory Committee on Pesticides. AChE activity was measured in peripheral tissues (nodose ganglia, left atrium, diaphragm, and quadriceps), red blood cells (RBC) and brain in male and female Beagle dogs (six months old, n = four/sex/group) exposed for six weeks to 0.0, 0.5, 1.0, or 2.0 mg/kg/day chlorpyrifos in their diets.

 

RBC AChE activity showed a slight (ca. 10%) but statistically significant decrease in the mid- and high-dose groups following the first day's exposure. RBC AChE activity reached significantly decreased (> 20% inhibition) steady-state levels in all groups within three weeks of treatment.

 

The small difference in brain AChE activity between high-dose dogs (male and female combined) and controls (approximately 7% inhibition) may represent a true effect, although it was not statistically significant and was not adverse « 20%). Differences in brain AChE activity between mid-dose males and controls were statistically significant (p < 0.05) but were not considered an effect of treatment in light of the lack of effect at this dosage in all previous dog studies and in females in this study. The NOEL for brain AChE was 1.0 mg/kg/day.

 

AChE activity in diaphragm, quadriceps, and nodose ganglia showed no significant differences between treated and control groups for males or females. However, it is possible that the lower activity in the left atrium AChE activity in high-dose male dogs was due to treatment, despite the lack of statistical significance and a comparable but opposite effect in female dogs. Therefore, the NOAEL for Beagle dog left atrium AChE was at least 1.0 mg/kg/day.

 

These results show that RBC AChE is more sensitive than peripheral tissue or brain to inhibition by chlorpyrifos. At the doses tested, there were no apparent differences in sensitivity of AChE of brain or peripheral tissues to inhibition by chlorpyrifos. On the basis of this study in Beagle dogs, the animal species considered most sensitive to chlorpyrifos-induced AChE inhibition, the acute NOAEL for RBC was 2.0 mg/kg body weight and the repeated-dose NOAEL for brain and peripheral tissues was at least 1.0 mg/kg/day.

Reference 9

Endpoint:

neurotoxicity: 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:

Pregnant Sprague-Dawley rats were exposed to chlorpyrifos (CPF) by gavage (in com oil) from gestation day (GD) 6 to lactation day (LD) 10. The purpose of the study was to 1) evaluate cholinesterase (ChE) inhibition at gestation day 20, postnatal days 1,5, 11,22 in dams and at gestation day 20, postnatal days 1,5, 11, 22, 65 in developing rats, and 2) to evaluate chlorpyrifos and its principal metabolites: chlorpyrifos-oxon (CPF-oxon), and 3,5,6-
trichloro-2-pyridinol (TCP) on gestation day 20, postnatal days 1, 5 and 11 in dams and pups, after dosing of the dams through part of gestation and lactation.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos
Lot MM930503-17; TSN 100227
Purity: 99.8%

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

gestation day (GD) 6 to lactation day (LD) 10

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

0.3 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:

Gestation day (GD) 20, Postnatal days (PND) 1,5 & II (N = 5 dams x 4 times = 20)

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

The dams were weighed on day 0 of gestation and daily beginning on day 6 of gestation through day 23 of gestation for dams that did not deliver a litter, or through postnatal day 10 for dams that did not deliver a litter.

Specific biochemical examinations:

Five dams, as well as 5 male and 5 female pups/dose level, were euthanized on gestation day 20 and postnatal days 1,5 and 11; samples of milk (dams) were taken for CPF, CPFoxon and samples of blood (dams and pups) were taken for CPF, CPF-oxon and TCP analysis.

Key result

Dose descriptor:

other: ChE levels of all tissues of high-dose pups rapidly returned to near control levels by PND5, further indicating a lack of differential sensitivity at this dosage level

Conclusions:

Thus, based on blood CPF levels, pups were more tolerant to ChE inhibition than were dams at these dosage levels. Inhibition of ChE occurred at all dosage levels in dams, but only at the high-dosage level in pups. At the high dose, ChE inhibition was greater in dams than in pups, and the relative degree of inhibition was RBC~plasma>heart>brain (least inhibited). Pup exposure via milk from dams given 5 mg/kg/day was roughly 120 µg/kg/day, which would be sufficient to inhibit blood ChE if pups were extra sensitive. Instead, ChE levels of all tissues of high-dose pups rapidly returned to near control levels by PND5, further indicating a lack of differential sensitivity at this dosage level.

Executive summary:

Pregnant Sprague-Dawley rats were exposed to chlorpyrifos (CPF) by gavage (in com oil) from gestation day (GD) 6 to lactation day (LD) 10. Dosages to the dams were 0, 0.3, 1.0 or 5.0 mg/kg/day. On GD 20, the blood CPF content in fetuses was about one half to one third the level found in their dams, indicating about a 2 to 3-fold level of maternal protection. Consistent with matenal:fetus differences in blood CPF, cholinesterase (ChE) inhibition occurred in dams at 0.3 mg/kg/day, but not in fetuses from dams given 1 mg/kg/day. Interestingly, no blood CPF could be measured (limit of detection 0.7 ng/g) in dams given 0.3 mg/kg/day, while fetuses of dams given 1 mg/kg/day had a blood CPF level of about 1 ng/g. Thus, based on blood CPF levels, pups were more tolerant to ChE inhibition than were dams at these dosage levels. Inhibition of ChE occurred at all dosage levels in dams, but only at the high-dosage level in pups. At the high dose, ChE inhibition was greater in dams than in pups, and the relative degree of inhibition was RBC~plasma>heart>brain (least inhibited). Pup exposure via milk from dams given 5 mg/kg/day was roughly 120 µg/kg/day, which would be sufficient to inhibit blood ChE if pups were extra sensitive. Instead, ChE levels of all tissues of high-dose pups rapidly returned to near control levels by PND5, further indicating a lack of differential sensitivity at this dosage level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

9 oral neurotoxicity studies were conducted on Rat, Dog and Hen. 13 week rat study conducted accorging to EPA FIFRA 82-7 is considered key for repeated dose cholinestarase activity.

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

neurotoxicity: acute inhalation

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:

This study was designed to provide essential data on the absorption and metabolism of inhaled particulate chlorpyrifos (CPF) aerosols and the kinetics of cholinesterase (ChE) inhibition resulting from inhalation exposure to CPF. The study was conducted in two phases. In phase 1 male and female rats were exposed to mean aerosol concentrations of 0, 13.3, or 66.7 mg/m3 CPF with an average aerodynamic particle size of approximately 1.4 µm (mass median aerodynamic diameter; MMAD). Serial sampling of blood and analysis of the RBC and plasma compartments for ChE activity indicated that inhalation of particulate CPF resulted in a concentration- and time-dependent inhibition of ChE activity with a peak of inhibition approximately 2 hours after the end of exposure. The phase 2 study was conducted using female rats (only) exposed to 0, 3.7, 12.9, 22.1, or 53.5 mg/m3 CPF with an average aerodynamic particle size of 1.8 to 1.9 µm MMAD.

GLP compliance:

yes

Specific details on test material used for the study:

Chlorpyrifos Technical
Lot# KC28161419; TSN101285
Purity: 99.8%

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Route of administration:

inhalation: aerosol

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2 µm

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 hours

Frequency of treatment:

once

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Phase I

Dose / conc.:

13.3 mg/m³ air (nominal)

Remarks:

Phase I

Dose / conc.:

66.7 mg/m³ air (nominal)

Remarks:

Phase I

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Phase II

Dose / conc.:

3.7 mg/m³ air (nominal)

Remarks:

Phase II

Dose / conc.:

12.9 mg/m³ air (nominal)

Remarks:

Phase II

Dose / conc.:

22.1 mg/m³ air (nominal)

Remarks:

Phase II

Dose / conc.:

53.5 mg/m³ air (nominal)

Remarks:

Phase II

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

A cage-side examination was conducted at least once a day.

Specific biochemical examinations:

ChE Activity in plasma, RBC, lung and brain and blood chlorpyrifos and chlorpyrifos-oxon concentrations were measured.

Key result

Dose descriptor:

NOEC

Remarks:

brain AChE

Effect level:

22.1 mg/m³ air (nominal)

Key result

Dose descriptor:

NOEC

Remarks:

RBC AChE

Effect level:

3.7 mg/m³ air (nominal)

Conclusions:

NOEC for CPF-induced inhibition of brain ChE activity was 22.1 mg/m3 (calculated absorbed dose = 2.2 mg CPF/kg body wt).
NOEC for RBC ChE inhibition = 3.7 mg/m3; calculated absorbed dose = 0.5 mg CPF/kg body wt

Executive summary:

This study was designed to provide essential data on the absorption and metabolism of inhaled particulate chlorpyrifos (CPF) aerosols and the kinetics of cholinesterase (ChE) inhibition resulting from inhalation exposure to CPF. The study was conducted in two phases. In phase 1 male and female rats were exposed to mean aerosol concentrations of 0, 13.3, or 66.7 mg/m3 CPF with an average aerodynamic particle size of approximately 1.4µm (mass median aerodynamic diameter; MMAD). Serial sampling of blood and analysis of the RBC and plasma compartments for ChE activity indicated that inhalation of particulate CPF resulted in a concentration- and time-dependent inhibition of ChE activity with a peak of inhibition approximately 2 hours after the end of exposure. The phase 2 study was conducted using female rats (only) exposed to 0, 3.7, 12.9, 22.1, or 53.5 mg/m3 CPF with an average aerodynamic particle size of 1.8 to 1.9µm MMAD.

 

Compared to ChE activity measured in control rats exposed to filtered air (0 mg CPF/m3 air), nose-only inhalation of particulate CPF resulted in a concentration- and time-dependent inhibition of ChE activity in RBC, plasma, and lung, but not in brain tissue. Under the conditions of this study, peak blood levels of CPF and oxon (Cmax) were observed during exposure while the time of maximum ChE inhibition (Tmax inhibition) occurred 2 hours or more after the end of exposure. Inhibition of plasma and lung ChE was statistically identified in all exposure groups while exposure-dependent inhibition of RBC ChE activity was statistically identified in only the three highest exposure groups (NOEC for RBC ChE inhibition = 3.7 mg/m3; calculated absorbed dose = 0.5 mg CPF/kg body wt). Exposure-dependent inhibition of brain ChE activity was statistically identified only in rats exposed to the highest (53.5 mg/m3 CPF) and was 3.5- to 6-fold less than that measured in RBC, plasma, and lung samples obtained from the same rats. Based on these data the NOEC for CPF-induced inhibition of brain ChE activity was 22.1 mg/m3 (calculated absorbed dose = 2.2 mg CPF/kg body wt).

 

This study will provide essential toxicokinetic data needed to refine parameter estimates based on inhalation of CPF and inform decisions to modify current PBPK/PD models that incorporate lung metabolism to permit better estimates of exposure/response relationships in humans.

Reference 2

Endpoint:

neurotoxicity: acute inhalation

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:

This study was designed to assess the effect of an acute 6 hour nose-only inhalation exposure to a saturated vapor concentration of chlorpyrifos on cholinesterase (ChE) activity in plasma, red blood cells (RBC), brain and lung and to determine the blood concentrations of the parent molecule, chlorpyrifos (CPF) and the metabolites chlorpyrifos oxon (oxon) and 3,5,6-trichloro-2-pyridinol (TCP) in CPF-exposed rats.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chlorpyrifos
Lot # 7299412; TSN100759
Purity: 97.6%

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

female

Route of administration:

inhalation: aerosol

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 hours

Frequency of treatment:

once

Dose / conc.:

0 mg/m³ air

Dose / conc.:

0.254 mg/m³ air

No. of animals per sex per dose:

8 control and 40 at 0.254 mg/m3. 8 test animals were sacrificed at 0, 2, 4, 6, and 12 hours following exposure

Control animals:

yes, concurrent vehicle

Observations and clinical examinations performed and frequency:

A cage-side examination was conducted at least once a day, preferably at the same time each day (usually in the morning).

A hands-on evaluation of skin, fur, mucous membranes, respiration and nervous system function (including tremors, convulsions, diarrhea and animal behavior) was conducted on all animals the day prior to exposure and on the 2, 4, 6, and 12 hours post-exposure sacrifice animals once following exposure.

All rats were weighed the day prior to exposure (test day -1). The 2, 4, 6, and 12 hours post-exposure sacrifice animals were also weighed once following exposure on test day 1.

Specific biochemical examinations:

Blood and tissues were isolated and processed to determine cholinesterase (ChE) activity in plasma, RBC, brain and lung tissue. Whole blood samples from n=4 rats in each experimental group were analyzed to determine the concentration of CPF, oxon and TCP. An additional group of unexposed control rats (n=8), that experienced the same laboratory and exposure tube acclimation and animal husbandry activities as the control and CPF exposure group animals, were sacrificed at the start of the air and CPF exposures. All blood and tissue analyses conducted on the nose-only exposed groups were also performed on rats in this unexposed control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical effects were noted during the six-hour exposure periods. In-life observations noted post-exposure were limited to soiling in four rats exposed to CPF. All rats appeared normal by test day 2.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Test day 1 post-exposure mean body weights were decreased 5.8% and 6.4% from test day -1 values for control and CPF exposed rats, respectively. Since similar decreases were observed in both control and CPF exposed rats, these losses are considered not to be treatment-related and are attributed to stress related to nose-cone confinement.

Description (incidence and severity):

No oxon greater than or equal to the lower limit of quantitation (LLQ; 0.00015 nmole/g blood) was measured in the blood of any control (unexposed or 0 ppb-exposed) or CPF-exposed rat at any time point. The maximum blood concentration of CPF was measured at the end of exposure. The concentration of CPF in blood was less than the LLQ (0.00015 nmole/g blood) by 6 hours post-exposure. The highest mean TCP concentration in blood was measured 0 and 2 hours after the end of exposure. Blood TCP levels decreased with time after exposure but were greater than the LLQ (0.089 nmol/g blood) through 12 hours after exposure. No inhibition of ChE activity was measured in plasma, brain, RBC or lung tissues, even in the presence of measurable systemic exposure to CPF. The results of this study indicate that acute inhalation exposures of up to 6 hours to a saturated vapor concentration of 0.254 mg/m3 (17.7 ppb) chlorpyrifos produce no discernable biological effects, including no clinical signs of exposure and no inhibition of ChE activity in plasma, brain, RBC or the portal of entry tissue, lung.

Key result

Dose descriptor:

NOEL

Effect level:

0.254 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects at highest dose tested

Conclusions:

Acute inhalation exposures of up to 6 hours to a saturated vapor concentration of 0.254 mg/m3 (17.7 ppb) chlorpyrifos produce no discernable biological effects, including no clinical signs of exposure and no inhibition of ChE activity in plasma, brain, RBC or the portal of entry tissue, lung.

Executive summary:

This study was designed to assess the effect of an acute 6 hour nose-only inhalation exposure to a saturated vapor concentration of chlorpyrifos on cholinesterase (ChE) activity in plasma, red blood cells (RBC), brain and lung and to determine the blood concentrations of the parent molecule, chlorpyrifos (CPF) and the metabolites chlorpyrifos oxon (oxon) and 3,5,6-trichloro-2-pyridinol (TCP) in CPF-exposed rats. Female CD(SD):Crl rats were exposed 6 consecutive hours to filtered air (0 ppb; control) or a time weighted average concentration of 17.7 ppb (0.254 mg/m³) CPF vapors using a flow-past nose only inhalation exposure system. This was the saturated CPF vapor concentration under the dynamic experimental condition on the day of exposure. The saturated vapor concentration was established by increasing the chlorpyrifos vapor concentration in the exposure chamber until the number concentration of aerosol particles in the CPF exposure atmosphere (25.85 particles/cm³) was greater than that in the 0 ppb control chamber (0.0609 particles/cm³). Oxon was present in the test material (<0.1%) and was also measured in the exposure atmosphere at a concentration of 0.003 mg/m³(0.24 ppb; ~ 1.4% of the mass concentration of CPF). Rats were sacrificed immediately (0 hr) and 2, 4, 6, or 12 hours after the end of exposure (n=8/exposure group/sacrifice time). Blood and tissues were isolated and processed to determine cholinesterase (ChE) activity in plasma, RBC, brain and lung tissue. Whole blood samples from n=4 rats in each experimental group were analyzed to determine the concentration of CPF, oxon and TCP. An additional group of unexposed control rats (n=8), that experienced the same laboratory and exposure tube acclimation and animal husbandry activities as the control and CPF exposure group animals, were sacrificed at the start of the air and CPF exposures. All blood and tissue analyses conducted on the nose-only exposed groups were also performed on rats in this unexposed control group. 

No oxon greater than or equal to the lower limit of quantitation (LLQ; 0.00015 nmole/g blood) was measured in the blood of any control (unexposed or 0 ppb-exposed) or CPF-exposed rat at any time point. The maximum blood concentration of CPF was measured at the end of exposure. The concentration of CPF in blood was less than the LLQ (0.00015 nmole/g blood) by 6 hours post-exposure. The highest mean TCP concentration in blood was measured 0 and 2 hours after the end of exposure. Blood TCP levels decreased with time after exposure but were greater than the LLQ (0.089 nmol/g blood) through 12 hours after exposure. No inhibition of ChE activity was measured in plasma, brain, RBC or lung tissues, even in the presence of measurable systemic exposure to CPF. The results of this study indicate that acute inhalation exposures of up to 6 hours to a saturated vapor concentration of 0.254 mg/m³(17.7 ppb) chlorpyrifos produce no discernable biological effects, including no clinical signs of exposure and no inhibition of ChE activity in plasma, brain, RBC or the portal of entry tissue, lung.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.254 mg/m³

Quality of whole database:

Two nose only inhalation neurotoxicity studies, one 2 hour and another 6 hours study was conducted in rats.

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Neurotoxicity of Chlorpyrifos was tested in rats under specific acute, subchronic, and developmental OECD neurotoxicity studies. It was also tested for delayed neuropathy in hens.

After single administration, Chlorpyrifos showed clinical signs from day 1 to 4, alteration in FOB parameters at day 1 (decreased activity and grip performance) at 50 mg/kg bw. Pathology/histopathology remained normal at sacrifice, but cholinesterase activity was not evaluated. So, the NOAEL was set at 10 mg/kg bw. In an independent study, Chlorpyrifos inhibited plasma, RBC, heart and brain cholinesterase activity at a single dose of 5 mg/kg bw, therefore, the NOAEL for cholinesterase inhibition was 1 mg/kg bw/day after a single dose of Chlorpyrifos.

After 13-week exposure to chlorpyrifos, female rats showed perineal soiling (5 mg/kg bw/day) at several weeks of the study and reduced motor activity in both sexes (15 mg/kg bw/day) at week 4, with no effects at 1 mg/kg bw/day. Cholinesterase activity was not evaluated in this study, however the study referred data of a previous subchronic study in which cholinesterase activity was evaluated. This subchronic study revealed that 1 mg/kg bw/day produced a significant RBC cholinesterase activity depression, therefore, the NOAEL for the 13-week neurotoxicity study was set at 0.1 mg/kg bw/day.

Chlorpyrifos showed no potential to produce delayed neuropathy after single or repeated administration.

Justification for classification or non-classification

Chlorpyrifos showed no potential to produce delayed neuropathy after single or repeated administration. Therefore, the substance does not need to be classified for neurotoxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.