Pirimiphos-methyl

Endpoint summary

Description of key information

- Oral: NOAEL = 0.4 mg/kg bw/day, male/female, rat, 90-day, equivalent to OECD 408, Clapp 1970

- Oral: NOAEL = 0.5 mg/kg bw/day, male/female, dogs, 2-year, equivalent to OECD 409, Rivett 1973

- Oral: NOAEL = 1 mg/kg bw/day, male/female, rat, 28-day, equivalent to OECD 407, Berry 1975

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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

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

Dose / conc.:

8 ppm

Remarks:

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

Dose / conc.:

80 ppm

Remarks:

Dietary equivalent to 4 mg/kg bw/day

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.

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.

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.4 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Pre-GLP study comparable to a guideline study, with restrictions.

System:

nervous system

Organ:

brain

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start: October 1979, experimental end: February 1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1978

Deviations:

no

GLP compliance:

no

Limit test:

no

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

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

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Pre-GLP study comparable to a guideline study, with restrictions.

System:

cardiovascular

Organ:

blood

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

90-day subchronic toxicity in rats, Clapp 1970

In a pre-GLP 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). 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.

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 week nine of treatment. The weight gain in all test groups was comparable to the control group, with the exception of the females in 80 and 360 ppm groups showing a reduced weight gain of 18 and 21 %, respectively, when compared to the control animals. The female rats in 80 and 360 ppm groups showed a reduced food utilization of 18 and 30 %, respectively, in comparison with the control. No adverse effects on the haematological parameters, gross or microscopic pathology were observed in treated animals. 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 the 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. No full recovery was after four weeks of treatment with normal diet. The male animals in 360 ppm group showed a tendency to inhibition of the cholinesterase activity, but this inhibition did not achieve statistical significance.

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

90-day subchronic toxicity study in mice, Martin & Atkinson 1996

The sub-chronic repeated dose toxicity of the test substance to female and male mice was studied in a 90-day dietary feeding study performed under GLP to OECD TG 408. Groups of ten female and male CD-1 mice were fed the test substance in the diet over a period of 13 weeks at concentrations of 0, 10, 30, 90, 270 and 810 ppm. 

Treating female and male CD-1 mice for 13 weeks with the test substance orally via the diet produced severe toxicity at the dose of 810 ppm, evident as clinical signs, weight loss and food consumption decreases. These effects were associated with marked depressions in plasma and RBC ChE activities in both sexes. The animals from this group were subsequently killed during week 2 of dosing. Marked depressions in plasma, RBC and brain ChE activities were evident at the time of necropsy. Less severe signs of clinical toxicity were observed at the treatment level of 270 ppm, and these appeared to be transient. At all tested concentrations, there were depressions in plasma and RBC ChE activities at most time points in both sexes. Brain ChE activity was depressed in the male 30 ppm treatment group, and in the male and female 90 ppm and 270 ppm treatment groups at necropsy in week 13. The 90-day NOAEL is considered to be 90 ppm in both sexes, based on the absence of adverse treatment related effects (e.g., clinical signs and decreases in body weight and food consumption).

2-year study in Beagle dogs, Rivett 1973

In a non-GLP compliant study which was similar to OECD 409 (pre-GLP and pre-guideline), beagle dogs (4/sex/dose level) received the test substance (in 0.1 mL corn oil via gelatine capsules) at 0, 0.5, 2 or 10 mg/kg bw/day for daily for 7 days a week for 2 years. Animals were observed for clinical signs, body weight, food consumption and water consumption. Ophthalmoscopy, haematology, clinical chemistry and urinalyses were performed pre-dosing and then every 3 months. Electrocardiograms were obtained pre-test and then every 6 months. Samples for plasma and erythrocyte cholinesterase determinations were taken pre-test, then weekly for 8 weeks, monthly to 6 months and then quarterly; brain cholinesterase activity was assayed in frontal cortex samples obtained at post-mortem. At termination, all animals received a gross examination, 14 organs were weighed, and over 30 organs and tissues together with any lesions were examined microscopically. 

Clinical signs of toxicity were mainly confined to the top dose group and included loose faeces and sporadic vomiting (within half an hour of dosing). Loss of appetite and body condition occurred from week 3 in 2 males at the top dose level and resulted in a loss of weight. One of the dogs showed improved bodily condition and rapid weight gain from week 7 onwards. The other dog showed no improvement until week 10, when appetite improved and there was subsequent gain of body weight. One top-dose female died on week 57 of the study; the cause of death was not evident. Body weight was reduced in top dose males from week 5 and from week 10 in males receiving 2 mg/kg bw/day; at the end of the study, body weight gains were similar in all male groups. In females, body weight was similar in control and top-dose animals, with low and mid-dose groups having greater body weight gain than controls. Food consumption was not markedly affected by treatment. Water consumption was higher in treated animals both before and during dosing but with no consistent dose response relationship. Ophthalmoscopic and electrocardiographic examinations revealed no abnormalities. Urinalyses and histopathological investigations showed no consistent findings. There is an indication of slight anaemia (~10% deficit in erythrocytes) in top dose animals but the biological significance of this is questionable given the large temporal and inter-animal variations.

There were no consistent treatment related effects other than those associated with cholinesterase inhibition. The inhibition of brain cholinesterase had a dose-response relationship and was statistically significant at all doses (males and females combined). The inhibition of brain cholinesterase only exceeded the 20% level that is considered to be adverse the top dose in males and females combined (10 mg/kg bw/day). At the two lower doses in males and the mid-dose in females, the mean value for inhibition of brain cholinesterase activity exceed the 20% level (≤25%), however when the intergroup variations (standard deviations) are considered there are overlaps between the values reported in the test and control groups and the effects are therefore not considered to be biologically relevant at ≤ 2 mg/kg bw/day in either males or females. Inhibition of erythrocyte cholinesterase activity was biologically and statistically significant at all time points in the top dose group and from week 25 in the mid-dose group (2 mg/kg bw/day); there were no test substance-related effects on erythrocyte cholinesterase activity at 0.5 mg/kg bw/day.

Based on the adverse effects of the test substance on plasma and red blood cell cholinesterase activity the NOAEL is 0.5 mg/kg bw/day.

28-day feeding study in rats, Berry 1975

In a non-GLP compliant study similar to OECD 407 (pre-GLP and pre-guideline), the test substance was administered via the diet to young 12 male and 12 female Wistar-derived rats at doses of 0, 5, 8, 10 and 50 ppm (dietary equivalent to 0.4, 0.8, 1 and 5 mg/kg bw/day) for a period of 28 days. All animals were examined daily for any abnormalities in behaviour or clinical condition and the following investigations were carried out: body weight, food consumption and cholinesterase activity. At termination of the study animals were euthanised and a post-mortem was carried out and any macroscopic lesions were noted.

There were no clinical signs of toxicity and no significant effects on body weight gain or food consumption, though food conversion efficiency was reduced (<10%) in top dose females.

Plasma cholinesterase activity was inhibited at the highest dose of 50 ppm test substance by 30% in male and 50% in female animals throughout the dosing period. No effect was detectable on erythrocyte cholinesterase activity. There were only sporadic minor changes in plasma cholinesterase activity at the 8 and 10 ppm levels unrelated to dosage. There were no notable macroscopic findings at autopsy.

It was concluded that the NOAEL for the test substance in the young rat was 10 ppm, dietary equivalent to 1.2 mg/kg bw/day.

Repeated dose toxicity: dermal

21 -day study in rabbits, Damment 1980

In a 21-day dermal study in rabbits, the repeated application of0, 4, 40 or 400mg/kg bw/day to rabbit skin under occlusive conditions elicited effects in all treatment groups. Mortalities were seen in 3 top dose animals, 1 mid-dose animal and 1 low dose animal but not in controls. Local irritation, confirmed microscopically was seen in all groups, but was more extensive at 400 mg/kg bw/day. Clinical signs were similar in all groups, but enteric discharge was more prevalent in top dose abraded females. There were no obvious or consistent treatment-related effects on body weight, food consumption, haematology or routine clinical chemistry findings; but marked temporal, inter-group and intra-group variations hinder assessment. Erythrocyte cholinesterase activity was clearly inhibited in top dose animals; with an indication of a dose-response relationship in females. Brain cholinesterase activity varied greatly between animals and groups though there is evidence of inhibition in top dose males from both the abraded and intact groups. With the exception of application sites, gross and microscopic findings were similar in all groups.

The overall NOAEL for systemic effects was considered to be 40 mg/kg bw/day.

Route-to-route extrapolation to the 3-month feeding study in rats will be performed for the long-term systemic DNEL for the dermal route since this study is more robust than the 21-day dermal toxicity study in rabbits.

Justification for classification or non-classification

The test substance has been studied in repeat dose oral studies in rats and dogs. The most significant toxicological effects are associated with inhibition of acetylcholinesterase. In assessing the toxicological significance of this effect, inhibition of erythrocyte and/or brain cholinesterase activity of ≥ 20% has been considered to be adverse. The data on the relative sensitivities of erythrocyte and brain acetylcholinesterase to the test substance are inconclusive (possibly owing to variable processing techniques), but support the use of erythrocyte and/or brain acetylcholinesterase as the primary determinant of adversity in the absence of either clinical signs or other evidence of toxicity. Therefore, on the basis of the finding of inhibition of brain and erythrocyte acetylcholinesterase activity (> 20 %) following oral administration of doses relevant for classification with the test substance is classified as STOT-RE 1, H372: Causes damage to organs (inhibition of acetylcholinesterase activity) through prolonged or repeated exposure, in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.