Paraquat-dichloride

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:

10 Sep 1985 to 2 Oct 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was designed to evaluate the potential subchronic toxicity of the test substance when administered dermally to albino rabbits for a period of six hours each day over an innterval of 21 consecutive days.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Paraquat-dichloride

EC Number:

217-615-7

EC Name:

Paraquat-dichloride

Cas Number:

1910-42-5

Molecular formula:

C12H14N2.2Cl

IUPAC Name:

1,1’-dimethyl-4,4’-bipyridyldiylium dichloride

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: eight to ten weeks
- Weight at study initiation: males, 1918 to 2383 grams; females, 1881 to 2318 grams
- Housing: Individual wire-bottom cage. Housing conditions will comply with the Animal Welfare Act (Publ. L 94-279) as set forth in 9 CFR, Part 3, and as outlined in the "Guide for the Care and Use of Laboratory Animals" (DHEW Publ. #NIH 80-23).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks prior to exposure period. Beginning the second week of acclimation, a plastic collar will be placed around each animal's neck so as to acclimate the animal to wearing it throughout the study. The collars will be used for the purpose of preventing the animals from ingesting residual test material.
- Justification for selection of species: The rabbit is an acceptable model for subchronic dermal toxicity studies under FIFRA, TSCA, and OECD guidelines. The rabbit is also sensitive to the toxic effects of a wide variety of compounds.
- Assignment to test groups: Animals will be randomly assigned to specific test groups on day 4 by a technique which minimizes mean body weight differences between groups.

DETAILS OF FOOD AND WATER QUALITY:
Food checked by supplier, water checked by laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 23.9
- Humidity (%): 50 to 86
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Remarks:

destilled

Details on exposure:

TEST SITE
- Area of exposure: Back
- Type of wrap if used: nonabsorbent binder to keep test material in contact with the skin. To prevent the ingestion of the test material, animals wore plastic collars for the duration of the study.
- Time intervals for shavings or clipplings: 24 hours prior to application of the control or test materials. Throughout the study, when needed (in most cases at leat once weekly).

REMOVAL OF TEST SUBSTANCE
- Washing : wet paper towels
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount applied: per animal 1.0 mL/kg bw
- Concentration:0.5, 1.15, 2.6, and 6.0 mg/kg bw test substance cation
- Constant concentration used: yes, based on body weight
- Vehicle: destilled water

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, animals wore plastic collars for the duration of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 5 g sample of the test materials was sent to the sponsor for analysis at the conclusion of the study. Additional samples of test material may be requested if analytical data warrant further evaluation.

Duration of treatment / exposure:

21 days

Frequency of treatment:

6 hours per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.5 mg/kg bw/day

Remarks:

Concentration of test substance cation.

Doses / concentrations 2

Dose / conc.:

1.15 mg/kg bw/day

Remarks:

Concentration of test substance cation.

Doses / concentrations 3

Dose / conc.:

2.6 mg/kg bw/day

Remarks:

Concentration of test substance cation.

Doses / concentrations 4

Dose / conc.:

6 mg/kg bw/day

Remarks:

Concentration of test substance cation.

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were selected with the intention of producing the following results:
(a) The highest dose level does not produce definite toxic effects but does not does not produce an incidence of mortality that would compromise evaluation of the study results, or is the highest dosage that can be applied.
(b) The intermediate dose levels produce minimal toxic effects, or are the dosage levels intermediate to the low and high dose levels.
(c) The lowest dose level does not produce any evidence of toxicity.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
Each animal was observed twice daily for mortality and moribundity. Daily cage side observations were conducted to determine any overt indications of a toxic effect potentially due to the test material. Thorough clinical evaluations were performed on days 1, 2, 4, 8, 11, 15, 18, and 21 prior to dosing.

DERMAL IRRITATION:
- Time schedule for examinations:
Dermal Irritation Scores were determined prior to dosing on days 1, 2, 4, 8, 11, 15, 18, and 21.

BODY WEIGHT:
- Time schedule for examinations:
Individual body weights were measured and recorded on days -7, -4, 1, 4, 8, 11, 15, and 18 prior to treatment, and at sacrifice.

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

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to study initiation and at the end of the study
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked in table were examined:
Erythrocyte Count
Total Leukocyte Count
Differential Leukocyte Count
Hematocrit
Hemoglobin Concentration
Platelet Count
Reticulocyte Count

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Prior to study initiation and at the end of the study
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table were examined.
Sodium
Potassium
Chloride
Calcium
Phosphorus
Uri C Acid
Blood Urea Nitrogen (BUN)
Creatinine
BUN/Creatinine Ratio
Total Protein
Albumin
Globulin
Glucose (Fasting)
B111rubin (Total and Direct)
Alkaline Phosphatase
Lactate Dehydrogenase (LDH)
Aspartate Aminotransferase (AST, SGOT)
Alanine Aminotransferase (ALT, SGPT)
Creatine Phosphokinase (CPK) Activity
Cholesterol
Triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: At study termination

Sacrifice and pathology:

GROSS PATHOLOGY:
A necropsy will be performed on all animals found dead or moribund during the course of the study and on all animals at the termination of the study. Each necropsy will include an examination of the external body surface and all orifices, the brain and cranial cavity. and all cervical, thoracic and abdominal viscera. The following organs will be trimmed and weighed as soon as possible after dissection;
Brain and entire brainstem
Liver Adrenals (Left and Right)
Kidneys (Left and Right)
Testes (Left and Right)
Ovaries (Left and Right)
lungs

Paired organs weighed separately.

HISTOPATHOLOGY:
Tissue Preservation: The following organs will be preserved in 10% neutral buffered formalin: lungs (Inflated with Formalin)
liver
Spleen
Adrenals
Brain
Heart
Kidneys
Gonads
Skin (Untreated Treated)
Gross Lesions

Histopathology: All of the tissues listed in this section from each test animal will be submitted for histopathological examination.

Statistics:

Upon completion of the study, all numeric data will be compiled into appropriate tables sequenced by test group. Means for each category for each test group will be compared statistically to the control values. The variance in body weights, organ weights, organ weights as a percentage of body weight, organ weight/brain weight ratios, and food consumption will be compared using Bartlett's test for homogeneity and a one-way analysis of variance. Group means will be compared using either Dunnett's test (homogeneous variance) or a modified t-test (non-homogeneous variance) to assess significance of differences.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All animals appeared normal throughout the course of the study.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

For both sexes exposure up to 1.15 mg/kg bw test substance ion were similar to the control. Two males, exposed to 2.60 mg/kg bw test substance ion, had small scabs on the back at days 18 and 21 and. One female within the same exposure group had small scabs at days 15, 18, and 21. Very slight erythema was observed for five males exposed to 6.00 mg/kg bw test substance ion at day 11, and on all males at day 15. Very slight to well-defined erythema was evident on all males at days 18 and 21. Very slight erythema was noted on four females, exposed to 2.60 mg/kg bw test substance ion, at day 11, and on five females at day 15. Very slight erythema persisted on four females until study termination, while one female had well-defined erythema at days 18 and 21. Small scabs were observed on the back of three 6.00 mg/kg bw test substance ion exposed animals (one male and two females) at day 11, and on all animals within the same exposure group at days 15, 18, and 21. Large scabs developed on the lateral and posterior area of the torso on two 6.00 mg/kg bw test substance ion exposed males and on three females.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Absolute body weight values at days -7, -4, 1, 4, 8, 11, 15, and 18 were similar for all groups for both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Both mean daily and total food consumption values were comparable in all groups of both sexes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical evaluation of pre-treatment haematology parameters revealed a significantly lower mean lymphocyte value for the females exposed to 2.60 mg/kg bw test substance cation and a significantly higher mean monocyte value for the females exposed to 0.50 mg/kg bw test substance cation. Both of these preinitiation differences were considered to be incidental in nature. All other pre-treatment mean clinical haematology values were similar.
Statistically significant clinical haematology findings at termination consisted of an incidental decrease in mean erythrocyte values for males exposed to 1.15 and 2.60 mg/kg bw test substance cation . All other clinical haematology parameters observed at termination were similar for all groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of the pre-treatment clinical chemistry mean values revealed significantly higher glucose values for the 5 females exposed to 6.00 mg/kg bw test substance cation.
Evaluation of days 22 and 23 clinical chemistry values revealed significantly higher albumin for females exposed to 1.15 mg/kg bw test substance cation. A positive trend was observed for the female groups for total protein, globulin, and aspartate aminotransferase values reflecting local dermal irritation. This change was considered incidental to compound application. All other clinical chemistry parameters determined at termination were comparable among the groups.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences or trends were noted in the terminal body weight values. Statistical evaluation of mean absolute organ weight values, mean organ/terminal body weight values, and mean organ/brain weight ratios, did not reveal any differences attributable to treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects involving the skin were observed in animals exposed to 6.00 mg/kg bw test substance ion of both sexes and included crusty scabs, redness, thickened appearance. and prominent subcutaneous vessels at the treatment site. Scabs over the treatment site were also noted for one female and two males exposed to 2.6 mg/kg bw test substance ion. Incidental findings included mottled lungs; dark focal areas in the lungs, kidneys, and stomach; pitted kidneys; pale focal areas in the heart and liver; small testes; ovarian cysts; and a few adipose masses.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the males, compound-related histopathologic changes were present in the treated skin in 5/6 animals exposed to 6.00 mg/kg test substance ion and 3/6 animals exposed to 2.60 mg/kg test substance ion. In the females, compound-related changes were present in the treated skin of 4/6 exposed to 6.00 mg/kg test substance ion. Morphologically these changes consisted primarily of epidermal erosion/ulceration, surface exudation, acanthosis and moderate to moderately severe chronic-active inflammation. In females exposed to 2.60 mg/kg test substance ion, the presence of compound induced lesions is equivocal. The mean scores for chronic-active inflammation and acanthosis were slightly higher than lower doses and controls. However, there was no evidence of erosion, ulceration, surface exudation or a markedly increased inflammatory response.
In the untreated skin in all groups and the treated skin exposed to 0 to 1.15 mg/kg test substance ion, the primary histopathologic alteration was minimal to slight chronic-active inflammation. This lesion was interpreted as a background finding with no differences between treatment and control groups.
There was no histologic evidence of systemic toxicity in tissues examined. Lesions present in all other organ systems were considered typical spontaneous/incidental findings associated with rabbits of this age and occurred without relation to treatment group. Many of these lesions were attributed to Encephalitozoon cuniculi.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Dermal irritation scores of exposure up to 1.15 mg/kg bw test substance were similar to the control for both sexes. Exposure to 2.6 mg/kg bw test substance ion resulted in small scabs on the back of two males at days 18 and 21 and for one female on days 15, 18, and 21. Very slight erythema was observed for five males exposed to 6.00 mg/kg bw test substance ion at day 11, and on all males at day 15. Very slight to well-defined erythema was evident on all males at days 18 and 21. Very slight erythema was noted on four females exposed to 6.00 mg/kg bw test substance ion at day 11, and on five females at day 15. Very slight erythema persisted on four females until study termination, while one female had well-defined erythema at days 18 and 21. Small scabs were observed on the back: of three animals exposed to 6.00 mg/kg bw test substance ion (one male and two females) at day 11, and on all animals within this exposure group at days 15, 18, and 21. Large scabs developed on the lateral and posterior area of the torso on two males and on three females exposed to 6.00 mg/kg bw test substance ion.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1.15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

original value presented in study

2

Dose descriptor:

NOAEL

Effect level:

1.59 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

3

Dose descriptor:

NOAEL

Effect level:

> 6 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

other:

Remarks:

original value presented in study

4

Dose descriptor:

NOAEL

Effect level:

> 8.29 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

other:

Remarks:

recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Calculation of key result

The doses of the test substance were expressed in paraquat ion, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 1.15 mg paraquat ion / kg bw = 1.59 mg pure paraquat dichloride / kg bw

(100/72.4) x 6.00 mg paraquat ion / kg bw = 8.29 mg pure paraquat dichloride / kg bw

Applicant's summary and conclusion

Conclusions:

In this 21-day repeated dose study, following dermal application of the test substance to rabbits, a NOAEL for dermal irritation of 1.15 mg/kg bw test substance ion (recalculated value: 1.59 mg/kg bw test substance) was found. No test substance-related systemic adverse effects were observed up to the highest dose tested 6.00 mg/kg bw test substance ion (recalculated value: 8.59 mg/kg bw test substance).

Executive summary:

The test material, was applied topically to the close clipped dorsal thorax of groups of six rabbits per sex at dosage levels of 1.5, 3.4, 7.8, or 77.9 mg/kg (0.50, 1.15, 2.60, and 6.00 mg test substance cation/kg, respectively) for six hours daily under an occlusive dressing. A similar group of rabbits were treated with 1.0 mL/kg of the vehicle, distilled water, and served as a control group. All animals were observed twice daily for overt toxic effects and given a thorough clinical examination which included an assessment of dermal irritation prior to dosing on days 1, 2, 4, 8, 11, 15, 18, and 21. Animals were weighed twice weekly and food consumption was measured weekly. Clinical pathology evaluations were conducted on samples collected prior to initiation of treatment and at termination. Following at least 21 days of treatment, animals were weighed, sacrificed, and given a complete gross necropsy. Selected organs were weighed and designated tissues were preserved, processed, and examined microscopically. Antemortem observations and clinical pathologic evaluations did not reveal any differences between the control and treated animals indicative of systemic toxicity. Slight to moderate dermal irritation was evident for all rabbits exposed to 6.00 mg/kg bw test substance ion except one female. Three rabbits exposed to 2.60 mg/kg bw test substance ion (two males and one female) also had signs of slight dermal irritation. Gross necropsy examination, analysis of the organ weight data, and microscopic examination of selected tissues also did not reveal any apparent evidence of systemic toxicity. Scabbing, redness, and/or thickening of the skin was noted at necropsy of those rabbits displaying dermal irritation during treatment. Likewise, microscopic examination revealed evidence of dermal irritation in 5 males and 4 females exposed to 6.00 mg/kg bw test substance ion and 3 males exposed to 2.60 mg/kg bw test substance ion. Findings included erosion/ulceration. surface exudation, acanthosis, and inflammation.