Diquat dibromide

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Aug 1985 to 05 Sep 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:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

1981

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

1992 + amendment 93/21/EEC (1993).

GLP compliance:

yes

Test type:

traditional method

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

Remarks:

CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult; 65-90 days (males), 66-91 days (females)
- Weight at study initiation: 313- 493 g (males); 197-314 g (females)
- Housing: Individually in wire-bottomed cages
- Diet: Purina Laboratory Rodent Chow #5001 ad libitum except during exposure.
- Water: mains water ad libitum except during exposure.
- Acclimation period: 21 - 46 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 22.6
- Humidity (%): 63 - 73
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 01 Aug 1985 To: 05 Sep 1985

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

water

Mass median aerodynamic diameter (MMAD):

>= 2.15 - <= 2.82 µm

Geometric standard deviation (GSD):

>= 1.95 - <= 2.05

Remark on MMAD/GSD:

Approximately 97 - 99 % of each aerosol was smaller than 10 microns

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Animals were exposed to aerosols of diluted test material. The generator solution was made by diluting 100 mL test substance to 1 L with distilled water. The test aerosols were generated using a nebuliser which pulled generator solution from a continuously stirred 1 L reservoir and unaerosolized material fromt he nebulizer drained back into the reservoir. Room air was drawn through the nebuliser to sweep the aerosol into the inlet at the top of the chamber at average flow rates of 4.6, 7.2 and 11.0 L/min, for the 0.15, 1.0 and 4.0 mg/L target concentrations, respectively. The nebuliser was operated at a pressure of 20 psi for the 2 lower target concentrations, and at 30 psi for the 4.0 mg/L concentration.
The exposure chambers were stainless steel, 27-inch, cubical chambers with pyramidal tops and bottoms and an internal volume of approximately 0.42 m3. The test and control rats were placed in 10 of 16 individual wire cages on one level within their respective chambers. Chamber pressure was maintained negative relative to the laboratory and the total flows through the test and control chambers were approximately 106 L/min. Exhaust from the bottom of each chambers passed through HEPA filters, a calibrated flow monitor and another filter train before discharge to the atmosphere. Control animals were exposed to filtered air only and were exposed concurrently with the 0.15 mg/L target exposure group. Chamber temperature, relative humidity and air flows were recorded initially and then every 30 minutes during exposure using an electronic thermometer, dial hygrometer and calibrated flow monitor, respectively.

TEST ATMOSPHERE
The total aerosol concentration of the test atmosphere, close to the animals’ breathing zone, was measured gravimetrically every 30 minutes during the exposure period. This was done by drawing the test atmosphere, at a known flow rate, for a known time, through a 25 mm diameter, Whatman GF/A filter housed in an open-faced filter holder. The amount of total aerosol was estimated gravimetrically for the 1.0 and 4.0 mg/L target exposures and with a Real-Time Aerosol monitor (GCA/Environmental Instruments) for the 0.15 mg/L target exposure. All filter samples were analysed for substance.

VEHICLE
The sample was tested as aerosols of test material diluted with distilled water.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

- Target concentrations: 0.15, 1.0, 4.0 mg/L
- Nominal concentrations: 0.16, 1.13, 3.86 mg/L

No. of animals per sex per dose:

5

Control animals:

yes

Remarks:

filtered air

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: following exposure, animals were observed twice daily, except at weekends when they observed once daily, during a 14-day observation period. The body weight of each rat was recorded before exposure and on days 2, 7 and 14 days following exposure.
- Necropsy of survivors performed: yes, external observation and examination of skin, spleen, pancreas, oesophagus, stomach, small and large intestine, liver, adrenals, kidneys, reproductive organs, bladder, heart, thymus, salivary glands, lungs, trachea, thyroid, brain, eyes and fat. Skulls, kidneys and a portion of liver were preserved in 10% neutral buffered formalin for possible future histological examination. Lungs were infused via the trachea, preserved in 10 % neutral buffered formalin and submitted for histopathological evaluation. Tissues were routinely processed, stained with haematoxylin and eosin and examined by light microscopy.

Statistics:

The LC50, slope and 95 % confidence limits were determined from the mortality data.

Results and discussion

Effect levels

Effect levels 1

Sex:

male

Dose descriptor:

LC50

Effect level:

0.121 mg/L air

Based on:

test mat.

95% CL:

>= 0.018 - <= 0.793

Exp. duration:

4 h

Remarks on result:

other: Original value presented in study

Effect levels 2

Sex:

female

Dose descriptor:

LC50

Effect level:

0.132 mg/L air

Based on:

test mat.

95% CL:

>= 0.022 - <= 0.781

Exp. duration:

4 h

Remarks on result:

other: Original value presented in study

Effect levels 3

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.125 mg/L air

Based on:

test mat.

95% CL:

>= 0.041 - <= 0.377

Exp. duration:

4 h

Remarks on result:

other: Original value presented in study

Effect levels 4

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

0.226 mg/L air

Based on:

test mat.

95% CL:

>= 0.034 - <= 1.481

Exp. duration:

4 h

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Effect levels 5

Sex:

female

Dose descriptor:

LC50

Effect level:

0.247 mg/L air

Based on:

test mat.

95% CL:

>= 0.041 - <= 1.459

Exp. duration:

4 h

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Effect levels 6

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.234 mg/L air

Based on:

test mat.

95% CL:

>= 0.077 - <= 0.704

Exp. duration:

4 h

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Mortality:

All animals exposed to 3.86 mg/L died 2-10 days after exposure. Three males and 2 females exposed to 1.13 mg/L died 3 - 5 days after exposure. There were no deaths in animals exposed to 0.16 mg/L.

Clinical signs:

other: See "Other findings: Other observations"

Body weight:

No statistical comparisons were made due to mortality and the different start dates of the exposures. Group mean body weights showed a weight loss in all exposed groups on day 2 after exposure (compared to day 0 before exposure). At the mid and high exposure levels, a continued weight loss was seen on day 7 but there was some weight gain by day 14 in males and females exposed to 1.13 mg/L.

Gross pathology:

At necropsy, lesions were seen in the lungs of all animals exposed to 3.9 mg/L and most of those at 1.13 mg/L. These lesions included reddening, darkening, oedema, mottling, and consolidations of the lung. In two males of the 3.86 mg/L group, yellow fluid was found in the trachea. No gross lesions were observed in the control and 0.16 mg/L dose groups.

Other findings:

HISTOPATHOLOGY
Histological examination of lungs revealed compound-related changes at all exposure levels, generally increasing in severity with increasing exposure concentration. All of the rats in the 1.13 mg/L and 3.86 mg/L groups had moderate to severe microscopic changes comprising subchronic inflammatory lesions; pulmonary congestion and oedema; and alveolar/septal thickening. Subchronic inflammation and/or alveolar/septal thickening were seen in three males and two females in the 0.16 mg/L group, but were of lower severity.

OTHER OBSERVATIONS
Squinted eyes and salivation were seen in exposed animas during exposure. Laboured breathing, abnormal respiratory sounds, increased respiratory rate, red oral /nasal /ocular discharge, reduced food intake and/or reduced faeces were seen following exposure. In rats exposed to 1.13 mg/L, there were also signs of slight hindquarter ataxia (2 males only), weakness (1 female only), unkempt appearance and sores or alopecia on the throat (seen in 2 males and 3 females between 6 - 9 days post exposure). At 0.16 mg/L, squinted eyes were present during exposure and salivation, red nasal and/or oral discharge, reduced faeces and unkempt appearance was present after exposure. Sores /alopecia on the throat was seen in 1 female 13 days post exposure but surviving animals appeared otherwise normal by 6 days post exposure.

Any other information on results incl. tables

Table 1: Mortality / animals treated

Group Number (5 rats/sex)	Dose level (mg/L)	Day of death	Number of deaths
			Male	Female
1	0	Total at day 14	0/5	0/5
2	0.16	Total at day 14	0/5	0/5
3	1.13	3	2	0
		4	0	1
		5	1	1
		Total at day 14	3/5	2/5
4	3.86	2	1	0
		3	2	0
		4	1	0
		7	0	1
		8	0	2
		9	0	2
		10	1	0
		Total at day 14	5/5	5/5

Table 2: Characteristic of test substance Atmospheres

Target Total Aerosol Concentration (mg/L)	Achieved Total Aerosol Concentration (mg/L)	Analysed test substance Concentration (µg/L)	MMAD(a)mm	GSD(b)
0	0		-	-
0.15	0.16	86.1	2.81, 2.82	1.98, 1.99
1.0	1.13	118	2.15, 2.21	2.01, 1.99
4.0	3.86	211	2.48, 2.44	1.96, 2.05

(a) Mass Median Aerodynamic Diameter(mm)

(b) Geometric Standard Deviation

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species):

The acute inhalation LC50 for male rats = 1.868 x 121 = 226 μg/L

The acute inhalation LC50 for male rats =1.868 x 132 = 247 μg/L

The acute inhalation LC50 for both sexes combined in rats = 1.868 x 125 = 234 μg/L

Applicant's summary and conclusion

Interpretation of results:

Category 2 based on GHS criteria

Conclusions:

The acute inhalation LC50 of test substance ion was 0.121 mg/L for males, 0.132 mg/L for females and 0.125 mg/L for both sexes combined. This is equivalent to an acute inhalation LC50 of 0.226 mg/L for males, 0.247 mg/L for females and 0.234 mg/L for both sexes combined of the test substance salt.

Executive summary:

In a GLP compliant acute inhalation toxicity study, comparable to OECD TG 403, groups of young adult Sprague-Dawley CD rats (5/sex/dose) were exposed by inhalation (whole body) for 4 hours, to aerosols of test substance in form of a technical concentrate (equivalent to 19.5 %w/w test substance ion), in distilled water, to target total aerosol concentrations of 0 (controls), 0.15, 1.0 or 4.0 mg/L. The amount of total aerosol was determined gravimetrically and all filter samples were analysed for test substance. The particle size distribution of the test atmosphere was analysed twice during the exposure period. After an observation period of 14 days after animals were necropsied. Specified tissues were submitted for histological examination. The average total aerosol concentrations were determined to be 0.16, 1.13 and 3.86 mg/L. The average mass median aerodynamic diameter (MMAD) of the aerosol ranged from 2.15 to 2.82 μm. Test substance ion concentrations for the exposures were determined to be 86.1 μg/L, 118 µg/L and 211 μg/L. The GSD (Geometric Standard Deviation) ranged from 1.99 to 2.05. Approximately 97 - 99 % of each aerosol was smaller than 10 microns.

Whole-body exposure for 4 hours to aerosols of diluted test substance, at average total aerosol concentrations of 0, 0.16, 1.13 or 3.86 mg/L, resulted in mortalities at 1.13 and 3.86 mg/L. All animals exposed to 3.86 mg/L died 2-10 days after exposure. Three males and 3 females exposed to 1.13 mg/L died 3 - 5 days after exposure. There were no deaths in animals exposed to 0.16 mg/L. Signs of toxicity included: squinted eyes and salivation (seen during exposure at all concentrations); laboured breathing, increased respiration rate, abnormal respiratory sounds; nasal, oral and/or ocular discharge; slight hindquarter ataxia / weakness; sores/alopecia on the throat; weight loss. At necropsy, treatment related lung changes were present in animals exposed to 1.13 and 3.86 mg/L. Histopathological examination revealed exposure-related, subchronic inflammation, congestion, oedema and alveolar/ septal thickening in the lungs of all rats exposed to 1.13 and 3.86 mg/L. Three males and two females in the 0.16 mg/L group had subchronic inflammation and alveolar / septal thickening of the lungs.

It is concluded that the acute inhalation LC50 of test substance was 0.80 mg/L for males, 1.09 mg/L for females and 0.97 mg/L for both sexed combined. This was equivalent to the following LC50 for the test substance ion: 0.121 mg/L for males, 0.132 mg/L for females and 0.125 mg/L for both sexes combined. And this is equivalent to an acute inhalation LC50 of 0.226 mg/L for males, 0.247 mg/L for females and 0.234 mg/L  for both sexes combined of the test substance salt.