Pirimiphos-methyl

Specific investigations: other studies

Administrative data

Endpoint:

specific toxicity, pathogenicity and infectiveness studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Start: 27 January 2012, end: 14 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency (2001). Office of Pesticide Programs (OPP) Guidance on Cholinesterase Measures in DNT and Related Studies

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

acute toxicity: oral

Test material

Test material information

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age/weight:
Part A F0 Generation Dams/F1 Generation Pups: Dams with litters, dams weighed 309.0-475 g on day after arrival. Pups age PND 5 or 8 on day of arrival.
Part B (subset 1) adult rats: Approximately 73-74 days / 318-359 g for males, 222-250 g for females at randomisation/study assignment.
Part B (Subset 2) F0 generation dams/F1 generation pups: Time-mated dams arrived at test facility on gestation day 18. Dams weighed 297.0-354.0 g day after arrival. Pups delivery naturally.
Source: Charles River Laboratories, Raleigh, NC, USA and Charles River Laboratories, Kingston, NY, USA.
Housing: Individually housed in stainless steel wire-bottomed cages (adults). Each dam and dam plus litter was individually housed in a nesting box.
Acclimatisation period: 3 days (dams and litters); 8 days (adults)
Diet: Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, MO, USA) ad libitum
Water: Local water that had been processed by passage through a reverse osmosis membrane ad libitum
Environmental conditions:
Temperature (target): 18-26°C
Humidity (target): 30-70%
Air changes: Minimum of 10
Photoperiod: 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared on the day before each dose administration. Dosing preparations were stored overnight in a refrigerator (2-8°C). The dosing formulations were stirred for at least 30 minutes before dosing and stirred continuously during dosing.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Single oral administration

Frequency of treatment:

Single dose

Post exposure period:

8 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Six per sex and dose (Part A, F1 generation neonatal pups)
Five per sex and dose (Part B, adult rats, F1 generation pups)

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 6 male and 6 female, PND 11 pups were dosed once, on PND 11, with pirimiphos-methyl in corn oil, at concentrations of 150 or 300 mg/kg bw. Two control groups of 4 male and 4 female pups were dosed with corn oil alone.
Groups of 5 male and 5 female, adult Crl:CD(SD) rats were dosed once, on Day 1, with pirimiphos-methyl in corn oil, at concentrations of 0 (control), 30, 100, 300 or 600 mg/kg bw.
Groups of 5 male and 5 female, PND 11 pups were dosed once, on PND 11, with pirimiphos-methyl in corn oil, at concentrations of 0 (control), 30, 100, 300 or 600 mg/kg bw.

Examinations

Examinations:

Observations:
(Part A) F0 generation dams/F1 generation neonatal pups: Dams were checked for viability twice weekly, clinical observations at least weekly and maternal behaviour daily. Litters were observed for dead pups at least twice daily. The pups were observed for general appearance daily and post-dose clinical observations were recorded approximately 1-2 hours after dosing and prior to the end of the normal working day.
(Part B) Adult rats (Subset 1): Rats were checked for viability at least twice daily and for general appearance twice during the acclimation period, on the day of dose administration and prior to euthanasia.
(Part B) F0 generation dams/F1 generation pups (Subset 2): Dams were checked for viability twice weekly, clinical observations at least weekly, parturition and maternal behaviour daily.

Bodyweight:
(Part A) F0 generation dams/F1 generation neonatal pups: Dams were weighed weekly. Pups were weighed on the day after arrival, on the day of dosing and daily during the post-dose period.
(Part B) Adult rats (Subset 1): Body weights were recorded twice during the acclimation period, on the day of randomization, on the day of dose administration, and prior to euthanasia.
(Part B) F0 generation dams/F1 generation pups (Subset 2): Dams were weighed at least once weekly. Pups were weighed on PND 0, 3, 6, and 9 and on the day of dose administration.

Cholinesterase evaluation:
(Part B) - Adult Rats (Subset 1): On day 1, approximately 8 hours after dosing, whole blood samples (3 mL) were collected from each rat. Blood samples were collected under isoflurane/oxygen anaesthesia from the inferior vena cava, prior to euthanasia. Following euthanasia, the brain was excised, weighed and homogenised. Blood and brain samples were held on wet ice or refrigerated until assayed for cholinesterase activity using a method based on Ellman’s method where one unit of ACHE will hydrolyze one micromole of ATC into choline and acetic acid per minute at pH 8.0, 37°C. Choline then reacts instantaneously with DTNB resulting in a yellow color. A standard curve is prepared with each standard having a different concentration of ACHE. A fixed amount of ATC and DTNB are added to the standards and unknowns. The rate of colour change (Vmax) is captured using a microtiter plate reader. The ACHE concentration in the unknowns is then back calculated on the standard curve using linear, unweighted regression.
(Part B) F1 generation pups (Subset 2): On PND 11, approximately 8 hours after dosing, whole blood samples (0.2-0.6 mL) were collected from each pup. Blood samples were collected via cardiac puncture immediately following ip injection of sodium pentobarbital. Following euthanasia, the brain was excised, weighed and homogenised. Blood and brain samples were held on wet ice or refrigerated until assayed for cholinesterase as described above.

Investigations post mortem:
Macroscopic examination: After collection of blood and brain, all adults and pups were discarded without further evaluation.

Organ weights: Brains were weighed at necropsy as described for cholinesterase evaluation.

Tissue submission: Brains were collected and evaluated as described for cholinesterase evaluation

Positive control:

Not applicable

Results and discussion

Details on results:

(Part A) F1 generation pups:
Mortality, clinical observations, body weight: There were no mortalities and no adverse clinical signs were observed in the male or female pups. Body weights were comparable among the male and female PND 11 pups on the day of dosing. In the male pups at 300 mg/kg bw, there was a test substance-related decrease in body weight gain observed on PNDs 11-12 and during the overall study period (PNDs 11-13) compared to control values. There was also a body weight loss at 300 mg/kg bw in the female pups on PNDs 11-12 and a decrease in body weight gain on PNDs 12-13 compared to control values. This resulted in an overall body weight loss for the female pups during the overall study period (PNDs 11-13). All rats gained weight at 150 mg/kg bw over the three days.

(Part B) Adult rats (Subset 1):
Mortality, clinical observations, body weight: There were no mortalities and no adverse clinical signs were observed. There were no effects on body weight.
Cholinesterase evaluation: RBC cholinesterase activity was statistically significantly reduced (p≤ 0.01 to p≤ 0.01) in the 100, 300 and 600 mg/kg bw dose groups in both the male and female rats in comparison with the vehicle control group values. There were no profound differences in the inhibition observed in the 300 mg/kg bw dose group as compared to the 600 mg/kg bw dose group in either sex. The reduction observed in both sexes at 30 mg/kg bw was considered minor and not toxicologically relevant. Brain cholinesterase activity was statistically significantly reduced (p≤ 0.01) in all substance-treated male rats at 100, 300 and 600 mg/kg bw and in female rats at 300 and 600 mg/kg bw as compared with the vehicle control group values.

(Part B) F1 generation pups (Subset 2):
Mortality, clinical observations, body weight: There were no mortalities and no adverse clinical signs were observed in the male or female pups. Body weights were comparable among the male and female PND 11 pups on the day of dosing.
Cholinesterase evaluation: RBC cholinesterase activity was statistically significantly reduced (p ≤ 0.01 to p≤ 0.05) in male and female pups in all dose groups administered the test substance as compared with the vehicle control group values. Brain cholinesterase activity was statistically significantly reduced (p ≤ 0.01) in all of the PND 11 male and female pups administered the substance as compared with the vehicle control group values.

Applicant's summary and conclusion

Conclusions:

It was determined that a dose level of 300 mg/kg bw would be used for adult rats and 100 mg/kg bw would be used for pups to establish the time-of-peak effect after an acute dose of the substance.

Executive summary:

The objective of this range-finding study was to collect data on the acute relative sensitivity of Crl:CD(SD) neonatal pups and adult rats to inhibition of cholinesterase activity after treatment with the substance in order to determine dose levels for future studies. Part A was to evaluate the sensitivity of Postnatal Day 11 (PND 11) pups to the substance and to establish doses for the PND 11 pups in the Part B portion of the study. Part B (Subset 1) was to measure cholinesterase activity in adult rats (at least 40 days of age) 8 hours following a single administration of pirimiphos-methyl. Part B (Subset 2) was to measure cholinesterase activity in PND 11 pups 8 hours following a single administration.
(Part A) F1 generation neonatal pups: There were no mortalities. There were no adverse clinical signs observed. There was a test substance-related decrease in body weight gain observed in the male pups administered 300 mg/kg bw on PNDs 11-12 and during the overall study period (PNDs 11-13). There was a body weight loss observed in the female pups administered 300 mg/kg bw on PNDs 11-12 and a decrease in body weight gain on PNDs 12-13, which resulted in a slight body weight loss in the female pups during the overall study period.
(Part B, Subset 1) Adult Rats: There were no mortalities. There were no adverse clinical observations observed. Statistically significant RBC cholinesterase inhibition was observed in both male and female rats at the 8 hour time point after an acute dose of 100, 300 and 600 mg/kg bw of the substance. Statistically significant brain cholinesterase inhibition was also observed in the male adult rats after an acute dose of 100, 300 and 600 mg/kg bw and in the female rats at 300 and 600 mg/kg bw of pirimiphos-methyl.
(Part B, Subset 2) F1 generation pups: There were no mortalities. There were no adverse clinical observations observed. Statistically significant brain and RBC cholinesterase inhibition was observed at 30, 100, 300 and 600 mg/kg bw of the substance.