Diquat dibromide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diquat dibromide

EC Number:

201-579-4

EC Name:

Diquat dibromide

Cas Number:

85-00-7

Molecular formula:

C12H12N2.2Br

IUPAC Name:

1,1'-ethylene 2,2'-bipyridyldiylium dibromide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Breeding Laboratories, Inc., Kingston, New York
Acclimation: 13 days
Age: 9 weeks (males), 10 weeks (females)
Weight: 256 to 297 g (males), 206 to 239 g (females)
Housing: individually in elevated stainless steel wire mesh cages
Feed: Purina Certified Rodent Chow #5002 ad libitum
Water: Elizabethtown Water Company drinking water ad libitum
Temperature: 68 to 76 °F
Humidity: 20 to 72%
Light: 12 hours light to 12 hours darkness cycle

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Prior to the application of the test substance, the fur of the back of test animals was clipped from an area measuring about 2.5 x 1.5 cm on each animal, using an electric clipper. Care was taken to avoid nicks or cuts during clipping. Animals were reclipped weekly or as necessary during the study. Plastic collars were applied during the acclimation period to allow time for the animals to adjust to wearing them. Collars were worn continuously by each animal for the duration of treatment to prevent ingestion of residual test material from the site of application.
A 1:4 dilution of the test material was prepared by adding 450 g of deionised water to 150 g of test material and gently agitating. Dose solutions adjusted by most recent body weight data were applied with a hand-held pipette or a microliter syringe (for the lowest dose). The test solution was applied to the mid-dorsal surface, in the center of the clipped skin area.
The test site was then covered with two layers of gauze dressing, followed with a wrapping of light plastic. Elastoplast was then wrapped over the plastic to prevent tearing. After six hours of exposure, the wrappings were removed, the residual test material rinsed from the skin with water and the skin wiped dry with soft paper towels.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

7 days/week for three consecutive weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

based on test substance cation species, 0.1 mL dose volume per kg bw

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

based on test substance cation species, 0.39 mL dose volume per kg bw

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

based on test substance cation species, 0.78 mL dose volume per kg bw

Doses / concentrations 4

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

based on test substance cation species, 1.55 mL dose volume per kg bw

No. of animals per sex per dose:

Six per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals considered suitable for the study were distributed randomly into five groups of six animals per sex.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

Animals were observed twice daily for mortality and clinical signs of toxicity. Detailed physical examinations for signs of toxicity and dermal evaluations were done during acclimation, before dosing and on day 2, 4, 8, 11, 15, 18 and 21 of dosing. Body weights were recorded during acclimation, immediately before first treatment and on days 3, 7, 10, 14, 17 and 21 of treatment. Food consumption was recorded weekly starting one week before start of treatment. Haematology and clinical chemistry evaluations were performed on all surviving animals at the terminal sacrifice. Urinalysis was performed for all animals in the control group and the group treated with 40 mg/kg bw/day.

Sacrifice and pathology:

Animals dying spontaneously, being killed in a moribund condition or sacrificed at the termination of the study were subject to a complete gross post-mortem examination. Organ weights were determined for adrenals, brain, kidneys, liver, testes with epididymides and ovaries. A selection of tissues was preserved and examined microscopically during histopathology.

Statistics:

Body weight, food consumption, haematology and clinical chemistry parameters, organ weights and organ/body and organ/brain weight ratios were analysed statistically. Mean values of all dose groups were compared to control at each time interval.
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, non-parametric procedures were used. The parametric procedures were standard one way ANOVA using F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a non-parametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.
A statistical test for trend in the dose levels was also performed. In the parametric case (i.e., equal variance) standard regression techniques with a test for trend and lack of fit were used. In the non-parametric case Jonckheere's test for monotonic trend was used.
The test for equal variance (Bartlett's) was conducted at the 1%, two-side risk level. All other statistical tests were conducted at the 5% and 1%, two-sided risk level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Common antemortem signs included hypothermia, hypoactivity, dyspnea, cyanosis, pale extremities, anogenital staining, little or no sign of stool, an apparent decrease in food consumption, general poor condition and an emaciated appearance.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The test substance caused dermal irritation, such as erythema, oedema, atonia and desquamation, as well as tissue destruction (necrosis, eschar formation) in several animals in all dose groups. The incidence and severity of the effects were generally dose-related. Severe necrotic effects were first seen after approximately one week of treatment. Affected animals in the group treated with 5 mg test substance cation species/kg bw/day exhibited transient necrosis and all animals in this group were free of significant irritation by day 18 of treatment. Necrosis in the animals treated at higher doses generally persisted throughout the study or until an animal's death.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female treated with 20 mg test substance cation/kg bw/day died. In the group treated with 40 mg/kg bw/day, one male and four females died. In the group treated with 80 mg/kg bw/day, five males and six females died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals which died showed substantial weight losses prior to death. The mean body weight in the group treated with 80 mg test substance cation/kg bw/day were markedly lower than control values from day 7 (males) or 10 (females) to study termination. Weight gains in males dosed at 5, 20 or 40 mg/kg bw/day were generally lower than gains in control animals. Mean weights for females treated at 5, 20 or 40 mg/kg bw/day were generally comparable to control weights.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in the groups treated with 40 and 80 mg cation/kg bw/day were lower than control values for males and females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The single surviving animal treated with 80 mg test substance cation/kg bw/day had slightly elevated haemoglobin, total erythrocyte and platelet values, possibly reflecting dehydration. Leukocytopenia was also seen in this animal at study termination. Haematology parameters in the other groups were not different from the control values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The single surviving animal treated with 80 mg test substance cation/kg bw/day had several abnormal clinical laboratory values suggestive of impaired renal, cardiac and hepatic function or general malnutrition and starvation. Abnormalities included elevated serum glutamic oxaloacetic transaminase, blood urea nitrogen (BUN) and uric acid values, an elevated BUN/creatinine value, evidence of electrolyte imbalance (elevated sodium, chloride and phosphorous levels, decreased potassium and calcium levels) and decreased serum albumin, total protein, cholesterol and triglyceride levels. With the exception of a markedly increased blood glucose value and decreased serum albumin and total protein values, relative to control levels, in one of the surviving females treated at 40 mg/kg bw/day, clinical chemistry parameters were unremarkable in all other surviving animals of the lower treatment groups.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight alterations in the single surviving male in the highest dose group were consistent with the markedly low terminal body weight of this animals and were similar to changes expected with food deprivation. No statistically significant differences occurred and no effects of test material administration were apparent in the other treatment groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross post-mortem examinations confirmed the dermal irritation and lesions seen during the study phase. Most of the abnormalities were observed in the treated skin of animals treated with 20, 40 and 80 mg test substance cation/kg bw/day (see information on dermal irritation for details). Gross findings in other tissues were few, occurred sporadically and were considered incidental.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All treated and untreated skin sections for animals from the controls and the groups treated with 40 and 80 mg test substance cation/kg bw/day and grossly abnormal skin sections from animals treated with 20 mg cation/kg bw/day were examined microscopically. An acute necrotising purulent dermatitis was the most prominent tissue alteration in the treated skin of animals of these treatment groups. The lesion was characterised by necrosis of the epidermis and papillary layer of the dermis with massive infiltrates of neutrophils. The viable epidermis was thick and hyperkeratotic. The epidermis immediately adjacent to the acute necrotising inflammation occasionally showed vesicle formation with neutrophils admixed with pink-staining vesicular fluid. The reticular layer of the dermis subjacent to the lesion contained dilated capillaries filled with erythrocytes and occasional fibrin thrombi. Acute vasculitis was seen in only one animal. Dermal hemorrhage was minimal, focal and rare. Affected adnexal structures such as the hair follicles and associated sebaceous glands showed various stages of degeneration. Inflammatory reactions around the hair follicles were either acute and purulent or granulomatous with multi-nucleated giant cell formation. Microscopic findings in other tissues occurred sporadically or with comparable incidences between the control and treated groups. No systemic effect of test material administration was apparent.

Histopathological findings: neoplastic:

no effects observed

Applicant's summary and conclusion

Conclusions:

Dermal administration of the test substance to albino rats for three consecutive weeks did not result in significant systemic toxicity. However, severe local dermal effects occurred leading to a moribund condition and subsequent death of most animals in the high-dose groups. A NOAEL for severe local dermal effects of 5 mg test substance cation species/kg bw/day, equivalent to 9.3 mg pure test substance, was determined following repeated dermal exposure for 6 hours per day, 7 days per week during three consecutive weeks.

Executive summary:

The repeated dose dermal toxicity of the test substance (tested as technical concentrate) to Sprague-Dawly CD rats was studied under GLP to EPA guideline 82-2. The substance was administered dermally, 7 days/week, for three consecutive weeks to 48 rats (six animals per sex per dose). The dose levels were 5, 20, 40 and 80 mg of test substance cation per kilogram of body weight per day (mg/kg bw/day). Twelve control animals (six per sex) were treated with the vehicle (distilled water) under similar conditions. After three weeks of treatment, all animals were sacrificed. The test substance produced dermal irritation (erythema, oedema, atonia and desquamation) and tissue destruction (necrosis, eschar formation) in several animals at all doses. Incidence and severity of these local effects were generally dose-related, but considerable variability among animals was seen. Severe necrosis effects were first noted in most animals after about one week of treatment. Affected animals exposed to 5 mg/kg bw/day exhibited transient necrosis and all animals were free of significant irritation on day 18 of treatment. Necrosis in affected animals treated with higher doses generally persisted throughout the study or until an animal's death, although no correlation between severe dermal effects and mortality was apparent. The dermal lesions were confirmed during necropsy. Microscopic abnormalities were observed primarily in the treated skin of animals treated at 20, 40 and 80 mg/kg bw/day, consisting of scabs, sores, desquamation, necrosis, exfoliation and fissures. Microscopic examination of grossly abnormal skne of treated animals revealed an acute necrotising purulent dermatitis in six of ten, eleven of twelve and twelve of twelve animals from the animals treated with 20, 40 and 80 mg/kg bw/day, respectively. Significant mortality occured in these treatment groups (one of twelve, five of twelve and eleven of twelve animals, respectively). Death was preceded in most animals by marked body weight losses, decreased food consumption and a variety of physical abnormalities (hypothermia, hypoactivity, anogenital staining, little or no stool, general poor condition, emaciated appearance). The single surviving animal in the group treated with 80 mg/kg bw/day exhibited a variety of haematologic and clinical chemistry abnormalities consistent with dehydration and malnutrition and suggestive of impaired renal, hepatic, or cardiac function at study termination. One of three surviving females treated with 40 mg/kg bw/day showed some clinical chemistry abnormalities, including elevated glucose value, decreased serum albumin and total protein values. No effect on clinical chemistry parameters was evident in the other animals treated with 40 mg/kg bw/day or in animal treated with lower doses. Organ weight data and microscopic examination of selected tissues revealed no evidence of systemic toxicity. Under the conditions of the study, a NOAEL for systemic toxicity following dermal administration of the substance to albino rats for three consecutive weeks was 40 mg test substance cation/kg bw/day, corresponding to 74.7 mg pure test substance /kg bw/day. However, the severe dermal local effects resulted in a moribund condition and the subsequent death of most animals in the high-dose groups. The NOAEL for dermal severe local effects was 5 mg test substance cation/kg bw/day, corresponding to 9.3 mg pure test substance/kg bw/day. Local dermal irritation occurred at this dermal dose, but the effects were transient and all animals were free of significant irritation at study termination.