Pirimiphos-methyl

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: October 1979, experimental end: February 1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1978

Deviations:

no

GLP compliance:

no

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 material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

This strain is the species recommended in the EPA test guideline. The strain was selected because it is readily available, is easy to handle, house, dose and bleed. There is a considerable amount of published toxicological information available on this species to assist in the assessment of the significance to man of any chemically induced changes.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Ranch Rabbits, Crawley Down, Sussex, England
Age: not specified
Weight: 2.25 to 2.9 kg (males), 2.18 to 2.9 kg (females); weights in additional control group were 2.86 to 3.0 kg (males), 2.95 to 3.17 kg (females)
Acclimatisation: 12 days

Housing: individually in grid floor cages in a single room with fan controlled air circulation
Temperature: 13 to 21 °C
Humidity: 30 to 80%
Lightning: photoperiod of 14 hours light to 10 hours darkness

Diet: Ranch Pellets, Grain Harvesters Ltd, Wingham, Canterbury, Kent ad libitum
Water: filtered mains tap water ad libitum

Administration / exposure

Type of coverage:

occlusive

Vehicle:

propylene glycol

Details on exposure:

The back and flanks of each animal were clipped free of hair using veterinary clippers 24 hours prior to first administrations. The treatment area was about 10% of the total body surface. It was reclipped at intervals to avoid that the regrowth of hair would have an effect on contact between test substance and skin. In additional groups, the treatment area was abraded once every seven days using a clipper head. The abrasion penetrated the horny layer of the epidermis but did not cause bleeding or damage to the underlying dermis.
Daily doses of the test substance or control substance were applied to the intact or abraded skin areas based on individual body weights.
A gauze pad (10 cm x 10 cm) was placed over the treated area and secured with a strip of impermeable adhesive plaster. This was held in place by a canvas jacket which was wrapped securely around the trunk of the animal. An Elizabethan collar was placed around the neck of each animal to prevent oral ingestion and interference with the wrappings. About six hours after the treatment the plasters and gauze were removed and the skin sites wiped to remove any test substance still remaining.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

3 weeks

Frequency of treatment:

Five days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five females, five males

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were allocated to treatment groups by means of a randomisation procedure based on body weight.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

Appearance, behaviour, general: all animals were observed at least once daily for overt signs of toxicity or behavioural change
Skin irritation: skin reactions were evaluated daily immediately prior to the application of the substance (erythema, oedema, desquamation not including eschar area, fissuring, atonia not including eschar area, eschar formation, exfoliation, folding)
Body weight: individual weights were recorded on the start of the study, then twice weekly and on the day of sacrifice
Food consumption: the total amount of food consumed per day was recorded daily
Haematology: a sample of blood (approximately 0.5 mL) was extracted from the marginal ear vein to perform haematological investigations
Clinical chemistry: a sample of blood (approximately 2 mL) was extracted from the marginal ear vein to perform analysis of blood clinical chemistry

Sacrifice and pathology:

The brain was divided into two equal portions at necropsy, and one side was processed for microscopic examination. The other side was homogenised to examine the cholinesterase activity.
Necropsies were carried out on all animals, which were painlessly killed by intravenous overdose of pentobarbitone sodium chloride. Major tissues and organs were examined for the presence of gross lesions. Organs were weighed prior to fixation (adrenals, brain, heart, kidneys, liver, ovaries, pituitary, testes, thyroids). A number of tissues and organs were fixed in 10% neutral buffered formalin (adrenals, brain, heart, kidneys, liver, ovaries, pituitary, testes, thyroids, treated skin, untreated skin, all unusual lesions). Samples of the tissues and organs were set in paraffin wax blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for microscopic examination.

Statistics:

Data were processed where possible to give dose and sex group mean values and standard deviations. Raw and processed data were examined for evidence of any toxicologically significant effects resulting from treatment. The study pathologist interpreted the macroscopic and microscopic pathology findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs periodically observed included lethargy, swelling of the abdomen, emaciation, enteric discharge, perianal staining and nasal and ocular discharges.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Skin irritation was observed in animals of all treatment groups. Animals treated at 400 mg/kg bw/day commonly showed moderate erythema, slight to moderate oedema and atonia and slight desquamation. Severe erythema and moderate desquamation were noted in some animals, but these observations were infrequent. In general, erythema was recorded throughout the study, oedema and atonia were recorded during weeks 2 and 3 and desquamation was recorded during week 3 only. Similar skin reactions were recorded within the high dose group for female and male animals and for animals with intact and abraded skin.
Skin reactions commonly described for animals treated at 40 and 4 mg/kg bw/day were slight erythema, atonia or desquamation. Animals treated at 40 mg/kg bw/day also occasionally showed slight oedema and moderate erythema (throughout the study), atonia or desquamation (during week 3 only). The skin reactions were similar for female and male animals and for animals with intact or abraded skin. Control animals showed slight erythema and desquamation periodically during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three animals in the group treated at 400 mg/kg bw/day, and one animal each in the groups treated at 40 and 4 mg/kg bw/day died early during the study. The deaths of the five animals were not considered related to treatment, but to respiratory and enteric disturbances associated with common laboratory diseases.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight changes of animals treated with the substance were, in general, similar to those of control animals throughout the study. However, females in the group of animals with abraded skin treated at 400 mg/kg bw/day had no body weight gains at all records after day 8 of the study compared to control animals or those in the other treatment groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The recordings of the food consumption indicated an equivocal effect of treatment on the consumption of food by male and female animals with abraded skin treated at 400 mg/kg bw/day. These animals consumed in general less food than the corresponding control animals or those in the other treatment groups.

Food efficiency:

not examined

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 erythrocyte and plasma activity of acetyl cholinesterase was depressed in blood samples taken from the animals treated at 400 mg/kg bw/day after 17 or 18 days of treatment. The pre-dose levels of the enzyme in this group were similar to both the pre-dose and terminal levels of the control groups or the other treatment groups being in the range of 147 to 265 iu/L (erythrocyte) and 291 to 413 iu/L (plasma). The terminal levels in the group treated at 400 mg/kg bw/day were noticeably lower in the range of 59 to 113 iu/L (erythrocyte) and 146 to 224 iu/L (plasma). No other dose-related changes occurred in any of the other clinical chemistry parameters examined. No significant effects on the activity of cholinesterase in the brain could be seen.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Propylene glycol (used as vehicle) produced minor skin reactions consisting of minimal to slight hyperkeratosis, acanthosis and hypergranulosis in the epidermis and a low grade of inflammatory reaction in the superficial dermis. The reactions to the test substance were of a similar type but slightly more extensive. The overall epidermal score for the three main parameters assessed was increasing in a dose-related manner. However, even at the highest dermal dose the epidermis was intact and showed only slight to moderate acanthosis, and the dermal inflammatory reaction was of a generally low grade.
Five animals died early in the study. Two of the three deaths in the group treated at 400 mg/kg bw/day were associated with poor respiratory conditions, but the cause of one death was not established. The two incidental deaths in the groups treated at 40 and 4 mg/kg bw/day were probably associated with enteric conditions.
Necropsy and histopathology findings in the surviving rabbits were generally infrequent and of a minor nature such as lymphoid or leucocyte foci in the liver and ectopic thymic tissue associated with the thyroid glands. There were no findings of any type or incidence to suggest any systemic, toxic effects related to the treatment with the substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy and histopathology findings in the surviving rabbits were generally infrequent and of a minor nature such as lymphoid or leucocyte foci in the liver and ectopic thymic tissue associated with the thyroid glands. There were no findings of any type or incidence to suggest any systemic, toxic effects related to the treatment with the substance.

Histopathological findings: neoplastic:

not examined

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity in a 21-day dermal repeated dose toxicity study was 40 mg/kg bw/day.

Executive summary:

The repeated dose toxicity of the substance following application to the skin of healthy male and female New Zealand White rabbits was studied in accordance with EPA Test Guideline 82-2. Three groups of 10 rabbits (5 per sex) with intact skin and three groups of 10 rabbits (5 per sex) with abraded skin were treated with the substance suspended in propylene glycol at doses of 4, 40 and 400 mg/kg bw/day. The substance was applied under occlusion (gauze pad fixed with impermeable plasters and held in place by a canvas jacket) for six hours per day on five days per week for a total period of three weeks. An Elizabethan collar was placed around the neck of each animal to prevent oral ingestion or interference with the wrappings. Following the six hour exposure period, the plasters and gauze were removed and the skin sites wiped to remove any remaining test substance.

Three animals in the group treated at 400 mg/kg bw/day, one animal in the group treated at 40 mg/kg bw/day and one animal in the group treated at 4 mg/kg bw/day died during the study. The deaths mostly occurred in the early phase of the study (between days 2 and 5) and were probably caused by spontaneous disease (e.g. poor respiratory or enteric conditions). However, the cause of the death of one animal on study day 11 could not be established. No adverse treatment-related clinical signs were noted during the study. Clinical signs indicative of common laboratory disease syndromes were observed in animals of all groups. Skin irritation consisting of erythema, oedema, desquamation and atonia was observed in animals of all treatment groups. These reactions were, in general, slight to moderate in animals treated at 400 mg/kg bw/day and slight in animals treated at lower levels. The body weight increases of some animals treated at 400 mg/kg bw/day were lower than those of control animals during the second and third week of the study. An equivocal depression in the food consumption was recorded for some animals treated at 400 mg/kg bw/day. No significant dose-related changes occurred in the haematology parameters examined. The activity of cholinesterase in erythrocytes and plasma was depressed in blood samples taken pre-terminally from animals treated at 400 mg/kg bw/day, but no significant effect on the activity of cholinesterase in the brain could be determined. No significant effect on organ weights was seen, and there were no gross or histological changes of any type or incidence to suggest any systemic toxicity of the test substance.