Diquat dibromide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

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 82-4 (90-Day Inhalation Toxicity)

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:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Inc., Kingston, New York
Lot no.: 288
Acclimation period: 13 to 15 days
Age: 57 days at study initiation
Weight: 165 to 195 g (males), 121-138 g (females)
Housing: individually in wire-bottom cages
Temperature: 20 to 24 °C
Relative humidity: 36 to 62%
Light: 12 hour light to 12 hour darkness cycle
Feed: Purina Certified Laboratory Rodent Chow Meal (#5002) ad libitum (except during exposure)
Water: East Bay Municipal Utility District drinking water ad libitum (filtered through 5 µm filter at the laboratory)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

water

Mass median aerodynamic diameter (MMAD):

>= 1.7 - <= 1.9 µm

Geometric standard deviation (GSD):

1.9

Remarks on MMAD:

MMAD was determined by means of cascade impactor samples

Details on inhalation exposure:

Stainless steel and glass rectangular chambers (Hazleton Systems, Aberdeen, Maryland, Model H1000) were used in the study. Chambers had a volume of about 1 m3. Chamber pressure was maintained slightly negative relative to the laboratory, and the total air flow through the test and control chambers was maintained at circa 14-16 air changes per hour (236 to 277 L/min). The supply air for the chambers was filtered and controlled for temperature. Exhaust from the bottom of each chamber passed through a HEPA filter, a calibrated flow monitor, and then through an activated carbon and another HEPA filter before discharge to the atmosphere. The animal caging within the chambers consisted of removable units with two rows of twelve individual cages per unit. Caging units were placed on the top and middle levels in each chamber and the lower levels were left empty. All three catch pans were in place.
Due to the slight variation in concentration within the chamber as determined in the pre-experiment distribution investigation, the study animals were assigned to a position within a caging unit and each unit was rotated between the top two chamber levels on a daily basis. The animals were transferred to the correct position in the exposure caging before each exposure. Following each exposure, each rat (including controls) was rinsed with warm water to remove test material which had deposited on the animal's fur (minimisation of oral exposure resulting from grooming).
Aerosols of diluted diquat concentrate were generated with an individual pneumatic nebuliser for each exposure concentration The aerosol for the high concentration was generated with Solo-Sphere nebuliser (Airlife Inc., Montclair, California). The nebuliser was operated with the aspirator control set at 100 (i.e. no dilution air through the nebuliser) and contained 400 mL of 5% test material solution (in distilled water) in its reservoir. The aerosol for the mid concentration was generated with an Ohio Ball-Jet nebuliser (Ohio Medical Products, Madison, Wisconsin) containing 300 mL of test material solution. The aerosol for the low concentration was generated with a 3-jet Collision modified MRE-type nebuliser (BGI, Incorporated, Waltham, Massachusetts) containing 150 mL of test material solution. Each nubuliser was loaded with freshly prepared solution before each exposure and was operated with filtered and dried compressed air at 10 psi. Nebuliser outputs were adjusted by controlling the flow of compressed air through the individual nebulisers. The approximate air flows used to generate the high, mid and low concentrations were 4.0, 4.5 and 3.0 L/min, which were measured with tapered glass rotameters. Each nebuliser was weighed before and after each exposure. The weight loss of the nubliser was divided by the total flow through the corresponding chamber to estimate the nominal concentration. The total air flow through the chambers was calculated from the hourly exhaust flow readings taken during the exposures.
The nebuliser outputs were directed via 1.3 cm diameter plastic pipe perpendicularly into the middle of the exposure chamber to supply air stream as it flowed through a stainless steel 5 cm pipe tee. The supply air, mixed with the aerosol, was routed to the exposure chamber via 5 cm diameter stainless steel flexible tubing. The aerosol entered the chamber at the center of the chamber top.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the study, the distribution of diquat cation within a chamber was tested at a concentration of approximately 5 µg/L. After 100 minutes of aerosol generation, samples were taken sequentially from five positions within the chamber above the top two cage units. Each position was sampled twice. Concurrently with each sampling a reference sample was taken from the location above the middle level within the chamber normally used for sampling. This indicated an average diquat concentration of 4.5 µg/L in the reference position and 5.3 µg/L in the five sampling positions. The coefficients of variation were 5.1% and 9.4% for the reference and sampling positions, respectively.
For each of the three exposure chambers used in the study to expose animals to the test substance, a Continuous Aerosol Monitor System (CAM, Process Particulate Monitors, Inc., Knoxville, Tennessee) was used to monitor the aerosol concentration in the chamber during exposure. The CAM system was a microprocessor-based electro-optical instrument consisting of individual scattered light sensors connected by cables to a central control/readout unit, which provided direct numerical readouts that could be related to aerosol concentration and printed time weighted average readings for each chamber every five or fifteen minutes. Each sensor was mounted to a 15 cm long 1.3 cm i.d. stainless steel sampling probe and sampled chamber atmosphere continuously at about 1 L/min. A sensor and probe were placed on each of the exposure chambers just above the middle level of caging at the front of the chamber. The CAM system was used for maintaining the desired aerosol concentration rather than providing definitive measurements of the aerosol concentrations.
For the definitive measurements, samples of chamber atmosphere were obtained during each exposure for analysis of the diquat cation. Samples were obtained by drawing atmosphere from each chamber through a 25 mm glass fiber filter (Whatman GF/A) in an open-face holder mounted on the end of a 15 cm long 0.85 cm i.d. stainless steel probe located above the middle level of cages at the rear of each chamber. The sampling flow was 2.0 L/min. Sampling times were 2 minutes for high, 10 minutes for mid and 15 minutes for low concentrations. Samples were taken every hour during the first five exposures and every other hour during the following ten exposures.
A sample from each test substance exposure chamber was taken weekly for particle size analysis, by drawing chamber atmosphere through a multi-jet cascade impactor (In-Tox Products, Albuquerque, New Mexico) at 20.0 L/min (same sampling times as for concentration determination).

Duration of treatment / exposure:

6 hours/day for three consecutive weeks

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.001 mg/L air (analytical)

Remarks:

pure test substance, recalculated value

Doses / concentrations 2

Dose / conc.:

0.002 mg/L air (analytical)

Remarks:

pure test substance, recalculated value

Doses / concentrations 3

Dose / conc.:

0.007 mg/L air (analytical)

Remarks:

pure test substance, recalculated value

Doses / concentrations 4

Dose / conc.:

0 mg/L air (analytical)

Remarks:

test substance cation species, original value given in the study

Doses / concentrations 5

Dose / conc.:

0.001 mg/L air (analytical)

Remarks:

test substance cation species, original value given in the study

Doses / concentrations 6

Dose / conc.:

0.004 mg/L air (analytical)

Remarks:

test substance cation species, original value given in the study

No. of animals per sex per dose:

10 rats per sex and dose

Control animals:

yes, concurrent no treatment

Details on study design:

The animals were randomly assigned to the four treatment groups so that differences in mean body weights between groups were minimised.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

All animals were closely examined daily for clinical signs of toxicity and the health status of all animals was checked in details on each morning prior to exposure. Body weights were measured on every animal before the first exposure and weekly thereafter. Food consumption and new food measurements were made weekly. An ophthalmologic examination was performed on each animal before the first exposure and at the end of the exposure period. A gross necropsy examination of all main study animals was done on day 21 or 22 and on day 42 for animals in the recovery groups. Blood was collected from all main study animals for haematology and serum chemistry investigations.

Sacrifice and pathology:

At scheduled termination, animals received an intraperitoneal anesthetic dose of sodium pentobarbital and were exsanguinated via the abdominal aorta. A selection of organs was examined for gross pathologic changes. Lungs, liver, brain, adrenals, testes and kidneys were weighed. A histophathologic examination of the nasal passages, trachea, lungs and any tissue with observed gross lesions from all animals was performed.

Statistics:

A computer program was used for statistical analyses of numeric results. Body weight, food consumption, clinical chemistry, haematology, organ weight and organ weight ratio data were analysed using a Bartlett's test for homogeneity of variances followed by an analysis of variance (ANOVA) when there were more than two groups of data. If the ANOVA result was significant (p <0.05), individual group comparisons were made with a two-sided Dunnett's test to identify statistically significant differences between groups. When only two groups were compared, a two-sided Student's t-test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Abnormal respiratory sounds in the high concentration group, increased incidence of anogenital discharge in the high concentration females. No clinical signs of toxicity were present in any high concentration animals by the end of the three-week recovery period, and all substance related effects were reversible.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of high concentration females and males was significantly reduced during and at the end of the exposure period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was significantly less than that of controls each week during the exposure period for high concentration females and males and for mid concentration males in the first exposure week.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean number of segmented neutrophils was significantly greater than in controls in low and high concentrations females and males. Furthermore, the mean number of lymphocytes was significantly less than in controls in the males of the low and high concentration groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slightly increased blood urea nitrogen concentration in mid and high concentration females, an increased alkaline phosphatase activity in high concentration females and a decreased cholesterol level in high concentration females was observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A compound-related and dose-related increase in lung weights of exposed animals was observed. For the males, the mean lung weight, lung to body weight ratio, and lung to brain weight ratio were significantly greater than controls for all exposed concentrations. Similar changes were observed for the mid and high concentration females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mottling and/or reddening of the lungs in all animals exposed to the substance (except low concentration males).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related changes occurred in the anterior nasal tissues of high concentration animals, consisting of epithelial dysplasia, erosion and chronic rhinitis; trace epithelial dysplasia in mid concentration groups. Exposure-related lesions in the lungs were observed in all animals exposed to the test substance. Multifocal chronic interstitial pneumonia and alveolar macrophages were present in males and females at all concentrations. Alveolar oedema was observed in the high concentration males and females and the mid concentration females. There was no hypertrophy or hyperplasia of Type I or Type II alveolar cells in any exposure group.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

LOAEC

Effect level:

0 mg/L air (analytical)

Based on:

act. ingr. (dissolved fraction)

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0 mg/L air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Due to increased lung weights and the presence of trace to mild lung lesions in male and female rats exposed to the lowest test concentration, a NOAEL could not be established in this 3-week repeated dose inhalation toxicity study. The LOAEL following three weeks of inhalation exposure was 0.5 µg test substance cation/L, equivalent to 0.93 µg pure test substance/L.

Executive summary:

The repeated dose inhalation toxicity of the substance to the rat was studied under GLP to EPA guideline 82-4. Groups of 10 male and 10 female Fisher 344 rats were exposed for 6 hours per day, 5 days per week for three consecutive weeks to chamber atmospheres containing an aerosol of test substance generated from diluted (5% v/v in distilled water) concentrate. The target concentrations of test substance cation were 0, 0.5, 1.0 and 4.0 µg/L. Additional control and high concentration groups of 10 rats per sex were held for a three-week recovery period. Chamber concentrations of substance cation were determined from air samples collected with glass fiber filters, and the overall average total concentrations were 0, 0.49, 1.1 and 3.8 µg test substance cation/L. Cascade impactor samples were obtained to estimate the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the aerosol. The average MMAD of the aerosols was 1.7 µm at the low and medium concentration and 1.9 µm at the high concentration. The GSD was 1.9 at each concentration and at least 99% of the aerosols were smaller than 10 µm. 

There were no exposure-related deaths during the study period. Animals exposed to the high concentration had abnormal respiratory sounds in both sexes and an increased incidence of yellow anogenital discharge in the females. Mean food consumption and body weights of male and female rats exposed to high concentrations were decreased throughout the exposure period. The mean body weight of high concentration females was not significantly different from controls during the three-week recovery period, but the mean body weight of high concentration males remained less than that of controls until sacrifice. No exposure-related changes in haematology or serum chemistry parameters were measured at the end of the exposure period. Significantly increased lung weights (asolute lung weight, lung to body weight ratio, lung to brain weight ratio) were occurred in a dose-dependent manner in low concentration males, mid and high concentration males and females at the end of the exposure period. At the end of the recovery period, the mean lung weights of the high concentration males were slightly greater than that of controls, but the differences were not statistically significant. Histopathologic evaluation indicated exposure-related changes in the anterior nasal tissues of some mid and high concentration animals. Exposure-related lung lesions such as multifocal chronic interstitial pneumonia and alveolar macrophages were observed in male and female rats of all dose groups. No exposure-related lesions were noted in other tissues and the nasal and lung changes were considered reversible within 21 days.

Since lung weights were increased and trace to mild lung lesions were present also in animals exposed to low concentrations, a NOAEL could not be established in this study, and the LOAEL for a three-week exposure period was determined as 0.5 µg test substance cation/L, equivalent to 0.93 µg pure test substance/L.