Pirimiphos-methyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1970

Report date:

1970

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

no

Remarks:

Study performed before GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pirimiphos-methyl

EC Number:

249-528-5

EC Name:

Pirimiphos-methyl

Cas Number:

29232-93-7

Molecular formula:

C11H20N3O3PS

IUPAC Name:

O-2-(diethylamino)-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate

Test animals

Species:

rat

Strain:

other: SPF

Remarks:

Wistar-derived

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: males: 268 - 367 g, females: 220 - 295 g
- Housing: housed five per cage in Wilmslow-type mobile rat units. The cages were constructed of 19 gauge galvanised wire mesh (1 cm2) on three sides and floor with a solid back, and measured 33 x 27.5 x 13.5 cm. They were suspended over collecting trays lined with absorbent paper and attached to each cage was a food hopper of capacity 300 g, and two water bottles each of capacity 225 mL.
- Diet: stock diet, ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:

oral: feed

Vehicle:

maize oil

Details on oral exposure:

DIET PREPARATION
All diets consisted of 77 parts of the stock diet, 18 parts of malt extract and 2 parts of maize oil with test substance, all by weight, to which was added 600 mL of water and the whole diet mixed mechanically for ten minutes after which it was moulded into pieces 3 - 6 cm in length and 1 cm diameter in a meat extruder. The experimental diets were identical with the control except that the appropriate quantities of the test material were incorporated into the diet before mixing.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 ppm

Remarks:

Dietary equivalent to 0.4 mg/kg bw/day in males and females

Doses / concentrations 2

Dose / conc.:

80 ppm

Remarks:

Dietary equivalent to 4 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

360 ppm

Remarks:

Dietary equivalent to 18 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

The animals on the various dose levels were introduced into the experiment over a period of two weeks, five animals from each group being introduced daily.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: at the beginning and at weekly intervals during the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY:
- Time schedule for collection of blood: pre-experimentally, at the mid point of the feeding study and immediately prior to killing the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: five males and five females per dose group
- Parameters checked: haemoglobin concentration, packed cell volume, mean corpuscular diameter, reticulocyte count, total and white cell counts, differential white cell counts, platelet counts and clotting function tests (prothrombin index and Kaolin/Cephalin time)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: once a week for five weeks pre-experimentally and then at one and two weeks after commencement of dosing followed by sampling at fortnightly intervals to the end of the experimental period
- Time schedule for collection of brains: at the end of dosing
- Animals fasted: Not specified
- How many animals: 5 rats from each group
- Parameters checked: erythrocyte, plasma and brain cholinesterase activity

Sacrifice and pathology:

GROSS PATHOLOGY:
At the end of the 90-day treatment period 20 males and 20 females from each group were killed with halothane and an immediate full post-mortem examination made. The remaining five male and five females in each group were treated in the same way after a four-week recovery period on normal diets. Organ weights were recorded and organ:body weight ratios calculated for five male and five female animals selected from each group. These calculations were made for liver, heart, lungs, adrenals, kidneys and spleen.

HISTOPATHOLOGY:
The following organs were examined: liver, kidney, spleen, heart, lung, adrenal, gonads, thymus, thyroid, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, salivary gland, brain (cerebrum, cerebellum and pons) and spinal cord.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All the animals survived the 90-day test period with the exception of one control male which died after four weeks from wounds received during fighting and one male on 360 ppm in the diet, which died of respiratory disease at nine weeks.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights show that the weight gain in all test groups is comparable with the control group, with the exception of the females in 80 and 360 ppm groups. These animals showed a reduced weight gain of 18 and 21 % respectively when compared with the control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The female rats in 80 and 360 ppm groups showed a reduced food utilisation of 18 and 30 % respectively when compared with the control value.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The activity of plasma cholinesterase was inhibited in 80 and 360 ppm groups to the extent of 40% in the males and 60% in the females in 80 ppm group, and 65% in the males and 80% in the females in 360 ppm, the inhibition becoming apparent after two weeks, but remaining constant to the end of the experimental period. The activities returned to normal within one week after cessation of dosing. No inhibition was seen at the lowest dose level (8 ppm). Inhibition of erythrocyte cholinesterase activity was seen in 360 ppm group only, and achieved 30% in the males and 50% in the females after two weeks on the test diets. This degree of inhibition persisted for the remainder of the experimental period and activity returned to normal during the four-week recovery period. Inhibition of brain cholinesterase activity occurred in the female rats of 80 and 360 ppm groups achieving a level of 40% in 360 ppm group. Recovery does not appear to be complete after four weeks. The male animals in 360 ppm group showed a tendency to inhibition of this activity, but this inhibition did not achieve statistical significance.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities attributable to the test material were seen. The male animals which died showed evidence of bleeding from the lacerations and bronchopneumonia in the lungs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

8 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

Remarks on result:

other:

Remarks:

Dietary equivalent to 0.4 mg/kg bw/day for both males and females

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

80 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 1.  Food consumption and body weight gain in female rats

Dose (ppm)	0	8	80	360	0	8	80	360
	Males				Females
Body weight gain
Starting weight	307.9	308.6	308.0	311.3	250.2	250.2	250.0	260.4
Terminal weight	437.2	437.4	446.4	439.3	285.3	285.3	278.8	288.0
Weight gain	129.3	128.8	138.4	128.0	35.1	35.1	26.8	27.6
Food consumption
Food consumption per rat (g)	1561	1487	1502	1520	1193	1201	1155	1216
Food intake per gram body weight (g)	12.1	11.6	10.9	11.9	34.0	34.2	40.1	44.1

 

Table 2. Lymphocyte counts following exposure to the test substance

Dose (ppm)	Time	Distribution 10^-3mm^3(mean ±SD)
		Males		Females
		Total WBC	Lymphocytes	Total WBC	Lymphocytes
0	Pre-exposure	8.32±0.59	6.45±0.55	6.68±1.23	5.73±1.14
	Mid-exposure	7.30±1.30	5.78±1.36	6.36±1.54	5.24±1.36
	Terminal	6.02±0.44	4.86±0.52	5.16±1.09	4.29±1.01
8	Pre-exposure	6.56±1.45	5.30±1.23	6.28±2.34	5.28±2.16
	Mid-exposure	6.62±1.85	5.69±1.59	6.14±1.27	5.29±1.52
	Terminal	5.46±1.53	4.39±1.48	5.12±1.50	3.96±1.45
80	Pre-exposure	6.86±2.39	5.59±2.00	6.78±1.79	5.69±1.42
	Mid-exposure	6.48±1.57	5.51±1.49	5.86±1.21	4.78±1.11
	Terminal	5.64±1.63	4.40±1.20	4.62±1.12	3.91±0.97
360	Pre-exposure	7.68±1.74	6.11±1.63	6.88±1.87	5.54±2.16
	Mid-exposure	5.78±1.95	4.72±1.41	6.04±1.61	4.76±1.49
	Terminal	6.02±1.57	4.77±1.70	4.30±0.83	3.42±0.63

 

Table 3. Cholinesterase activities (means, absolute and % inhibition) in rats (~5/group) fed diets containing the test substance.

Week	1 pre-	1	2	6	12	1 recovery	4 recovery
Dose (ppm)
Erythrocyte
Males      0^£	1.0	1.0	0.9	1.4	1.3	0.9	1.1
8 (% inhibition^§)	+27	+20	+11	5	+8	+31	+20
80 (% inhibition^§)	+31	+6	+10	21	+11	+13	+5
360 (% inhibition^§)	+17	+8	34	60	11	34	+16
Females    0^£	1.2	1.0	1.2	1.4	1.2	1.0	1.1
8 (% inhibition^§)	1	+28	3	5	14	+11	+4
80 (% inhibition^§)	14	+35	1	24	22	6	+3
360 (% inhibition^§)	18	5	48	51	60	29	22
Brain
Males     0^#	N.P.	N.P.	N.P.	N.P.	30.6	N.P.	30.5
8 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	+3	N.P.	+2
80 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	1	N.P.	+9
360 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	12	N.P.	14
Females   0^#	N.P.	N.P.	N.P.	N.P.	31.8	N.P.	31.4
8 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	6	N.P.	+1
80 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	20	N.P.	21
360 (% inhibition^§)	N.P.	N.P.	N.P.	N.P.	42	N.P.	35

N.P. - not performed

^£-umoles/ml/min

^#- delta pH/g/h

^§compared with control values rather than pre-test values for the group

+ increase in cholinesterase activity compared with the control

Applicant's summary and conclusion

Conclusions:

The NOAEL is 8 ppm, dietary equivalent to 0.4 mg/kg bw/day, based on brain cholinesterase activity inhibition in female rats.

Executive summary:

In a non-GLP compliant study which was similar to OECD 408 (pre-GLP and pre-guideline), groups of 25 male and 25 female SPF Wistar-derived, pathogen free rats were maintained for 90 days on diets containing 0, 8, 80 and 360 ppm test substance (dietary equivalent to 0.4, 4, 18 mg/kg bw/day). Animals were observed for clinical signs, body weight, food consumption. Blood samples for haematology were taken from 5/sex/group pre-dosing, mid-term and at week 12. Plasma and erythrocyte cholinesterase activities were determined pre-dosing, at weeks 1, 2, 4, 6, 8, 10 and 12 and at weeks 1 and 4 of the recovery period. Brain cholinesterase measurements were performed at terminal sacrifice. At the end of the 90-day treatment period 20 males and 20 females from each group were killed with halothane and an immediate full post-mortem examination made. The remaining five male and five females in each group were treated in the same way after a four-week recovery period on normal diets. Organ weights were recorded and organ:body weight ratios calculated for five male and five female animals selected from each group. These calculations were made for liver, heart, lungs, adrenals, kidneys and spleen.

All the animals survived the 90-day test period with the exception of one control male which died after four weeks from wounds received during fighting and one male on 360 ppm in the diet, which died of respiratory disease at nine weeks. The mean body weights show that the weight gain in all test groups is comparable with the control group, with the exception of the females in 80 and 360 ppm groups. These animals showed a reduced weight gain of 18 and 21 % respectively when compared with the control animals. The female rats in 80 and 360 ppm groups showed a reduced food utilization of 18 and 30 % respectively when compared with the control value. No adverse effects were observed in the haematological parameters, gross or microscopic pathology. The activity of plasma cholinesterase was inhibited in 80 and 360 ppm groups to the extent of 40% in the males and 60% in the females in 80 ppm group, and 65% in the males and 80% in the females in 360 ppm, the inhibition becoming apparent after two weeks, but remaining constant to the end of the experimental period. The activities returned to normal within one week after cessation of dosing. No inhibition was seen at the lowest dose level (8 ppm). Inhibition of erythrocyte cholinesterase activity was seen in 360 ppm group only, and achieved 30% in the males and 50% in the females after two weeks on the test diets. This degree of inhibition persisted for the remainder of the experimental period and activity returned to normal during the four-week recovery period. Inhibition of brain cholinesterase activity occurred in the female rats of 80 and 360 ppm groups achieving a level of 40% in 360 ppm group. Recovery does not appear to be complete after four weeks. The male animals in 360 ppm group showed a tendency to inhibition of this activity, but this inhibition did not achieve statistical significance.

Under the conditions of this study, the NOAEL is 8 ppm, dietary equivalent to 0.4 mg/kg bw/day, based on brain cholinesterase activity inhibition in female rats.