Paraquat-dichloride

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sprague-Dawley CD rats were exposed to various concentrations of test substance ion aerosols (0.01, 0.10 and 0.50 µg/L), six hours a day, 5 days a week for 3 weeks (total 15 exposures) to determine the following:
1) Whether test substance does attain a steady state concentration in rat lung following repeated exposures at 0.01, 0.1 and also 0.5 µg/L test substance ion.
2) The elimination half-life of test substance in rat lung at different exposure levels and after 1 and 15 exposures.
3) Whether there is a sex difference in lung test substance levels after repeated exposures to test substance aerosols.

This study was performed using a whole-body exposure method in which 85 male rats and 10 female rats were exposed per treatment dose. For the control group, 10 male rates were used. The analysis of test substance ion concentration in the lungs and kidneys were used as reference point for the treatment groups.
The control group was sacrificed on the first day of exposure.
For objective 1, male rats were sacrificed on day 1, 2, 3, 4, 5, 10 and 15 and 5 female rats from each group were on day 5 and 15 for analysis of lung and kidney test substance content.
For objective 2, male rats were sacrificed 7 hours after exposure, on day 1, 2, 3 and 6 for analysis of lung and kidney test substance content.
For objective 3, the results from the female rats were compared to the data obtained from male rats.

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:

rat

Strain:

Sprague-Dawley

Remarks:

CD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 26 to 30 days old
- Weight at study initiation: males 116 to 136 g, females 114 to 146 g
- Housing: The animals were housed 4 or less of like sex to a cage on open, relocatable racks in the exposure/holding rooms. All animals in the same group were housed on the same rack. The rat cages were 56 cm x 38 cm x 18 cm and were constructed of polypropylene with a polished stainless steel mesh base and top. Each cage was fitted with a polythene bottle with a stainless steel nozzle, for water. A plastic tray was placed under each cage to catch excreta. The tray contained a sheet of absorbent paper which was changed daily.
- Diet: laboratory diet, ad libitum (not during exposure)
- Water: tap water, ad libitum (not during exposure)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 ± 5
- Humidity (%): room 1: 58 (SD = 4.6). room 2: 57 (SD = 5.5)
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

< 0.7 µm

Remarks on MMAD:

The mean percentage (standard deviation) of particles of less than approximately 0.5 µm in aerodynamic diameter were 87 % (SD 5.9, N = 14), 65 % (SD 3.7, N = 15) and 39 % (SD 4.7, N = 15) for low, intermediate and high dose atmospheres respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposures were carried out in chambers constructed from stainless steel and glass. The aerosol was fed into the main chamber air supply through a flexible plastic tube. The chamber bodies were 182 cm wide, 152 cm high and 189 cm deep and the internal volume was approximately 6 m3.
- Method of holding animals in test chamber: During exposures the rats were placed in stainless steel mesh cages of height 18 cm and base dimensions 54 x 28 cm. Each cage was subdivided into 4 equal sized compartments by vertical stainless steel mesh partitions. One rat was placed in each compartment. Cages were placed on 2 levels in the exposure chamber.
- System of generating aerosols: Aerosols of the test substance was produced by ultrasonic nebulisation of a solution of the test substance (technical liquor) in distilled water. The aerosol was fed into the supply air to the exposure chamber. In this device vibrational energy is transmitted by means of a couplant fluid and diaphragm to the solution in the nebuliser chamber. The solution is atomised into minute droplets and the aerosol carried from the nebuliser chamber in a continuous airstream.
- Temperature, humidity in air chamber: mean temperature per chamber between 23.3 and 23.8 ˚C (standard deviations per chamber between 0.78 and 0.98). Mean relative humidity per chamber between 70 and 79 % (standard deviations per chamber between 5.8 and 6.4%).
- Air flow rate: 1500 L/min
- Method of particle size determination: Particle size estimations were carried out using a cascade impactor. This device comprises a system of 4 air jets impinging, in series, on glass discs and passing finally through a filter which may be inserted after the fourth disc when required. Based on the diameter of the aerosols, the retention disc will collect these particles if they are too big to pass the disc. Based on the quantity of aerosols present on the disc after testing, the aerosol diameter was determined.
- Exposure regime: The exposure regime consisted of a 6 hour exposure on each of 15 days during a 3 week period. The first exposure was carried out on a Friday. Exposures were then carried out on Monday to Friday of the following 2 weeks and Monday to Thursday of the final week. Exposures were carried out between 9:30 a.m. and 4:40 p.m. The beginning of the 6 hour exposure was measured from the time at which the exposure chamber was estimated to have reached 90% of final atmosphere concentration. This was estimated to be 10 minutes after the nebuliser had been switched on. The nebuliser was therefore switched on for a period of 6 hours and 10 minutes. At the end of the exposure session the animals were left in the exposure chamber for 15 minutes before being returned to their holding cages. No food or water was available during exposures. The position of the exposure cages within the exposure chamber was altered daily according to a pre-set pattern to minimise any possible effects of cage position within the chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test substance ion in the chamber atmosphere was determined by drawing air from the chamber through a glass microfibre filter at a rate of 40 L/min for a time dependent on the concentration being analysed. The sampling times were 2, 10 and 40 minutes for high, intermediate and low dose chambers respectively. The filter was incubated with saturated ammonium chloride solution following sodium dithionite in N sodium hydroxide. Absorbance at 600 nm was used as quantitative measure using the standard curve from the analytical standard to determine the test substance concentrations.

- Samples taken from breathing zone: no, however, the distribution of the aerosols in the exposure chamber was tested and considered to be acceptable for an aerosol.

VEHICLE
- air, but specifics not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test substance ion in the chamber atmosphere was determined by drawing air from the chamber through a glass microfibre filter at a rate of 40 L/min for a time dependent on the concentration being analysed. The sampling times were 2, 10 and 40 minutes for high, intermediate and low dose chambers respectively. The filter was held in an "open face" filter holder, which was fitted into a sampling port on the chamber. Following sampling, the filter was placed in 10 mL of saturated ammonium chloride solution and shaken. 2 mL of a 1 g% solution of sodium dithionite in N sodium hydroxide was then added. The sodium dithionite solution was prepared freshly at 2 hour intervals. The resultant blue solution was then filtered into a disposable cuvette and the absorbance at 600 nm read against an ammonium chloride/sodium dithianite blank. The amount of test substance present in the sample was then determined from a standard curve and the chamber aerosol concentration calculated from the volume of air sampled and the amount of test substance ion present. In separate experiments the recovery of test substance from such filters was determined and a correction factor included in the calculations to account for this, The chemical analysis technique was according to the method supplied by the Sponsor.

Duration of treatment / exposure:

Total 15 exposures

Frequency of treatment:

Six hours a day, 5 days a week for 3 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.01 mg/m³ air

Remarks:

Concentration of test substance cation.

Doses / concentrations 2

Dose / conc.:

0.1 mg/m³ air

Remarks:

Concentration of test substance cation.

Doses / concentrations 3

Dose / conc.:

0.5 mg/m³ air

Remarks:

Concentration of test substance cation.

No. of animals per sex per dose:

Control group: 10 male rats, no females
Treatment groups: 85 males, 10 females

Control animals:

other:

Details on study design:

- Dose selection rationale: A previous inhalation study established the level of test substance present in the lung of rats exposed to aerosols of test substance (0.01 μg/L test substance ion and 0.1 μg/L test substance ion) on 5 or 15 occasions during a three week period. The results suggested that there was no increase in the lung concentration of test substance beyond 5 exposures and that the elimination half-life from the lung after 15 exposures was approximately 2 days. It was concluded that there was no sex difference in lung test substance levels although the results after 5 exposures showed that the level of test substance in the lungs of female rats was higher than in males.
- Study design can be found in 'Any other information on materials and methods incl. tables'.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily throughout the study.

Sacrifice and pathology:

None

Other examinations:

The control group was sacrificed on the first day of exposure. Lung and kidneys were used for analysation of test substance content.
For objective 1, male rats were sacrificed on day 1, 2, 3, 4, 5, 10 and 15 and 5 female rats from each group were on day 5 and 15 for analysis of lung and kidney test substance content.
For objective 2, male rats were sacrificed 7 hours after exposure, on day 1, 2, 3 and 6 for analysis of lung and kidney test substance content.

Statistics:

The logarithms of test substance concentration in the lungs of rats killed immediately after exposure was considered for the different numbers of exposures by the analysis of variance separately for each dose group. The differences amongst the means after different numbers of exposures were investigated using the Student's 't'-test.
For rats killed at different times after 1 or 15 exposures the lung test substance concentrations were analysed to estimate the elimination half-lives. The mean of the logarithms of paraquat concentration in the lungs at each time point were calculated and for each dose and number of exposures (1 or 15) a straight line was estimated for these means against time. The slope of this line was used to estimate the elimination half-life.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the 2-day period between the 6th and 7th exposures, pilo-erection, tachypnoea and brown nasal staining were observed in the majority of rats exposed to 0.50 µg/L test substance ion. One rat exposed to 0.01 µg/L test substance, was found to have a discrete subcutaneous nodule approximately 1.5 cm diameter behind the right forelimb. This was first noted following the 14th exposure and, when the animal was killed, the nodule was preserved in 10 % buffered formalin but not examined.
No clinical abnormalities were observed in any of the other groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The only deaths which occurred were rats exposed to 0.50 µg/L test substance ion. One female and 12 male rats died during the 2-day period between the 6th and 7th exposures and 1 male rat died 2 days after the 15th exposure. There were no deaths in any of the other groups.

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Kidney:
Since the kidney is the organ responsible for the excretion of test substance the concentration of test substance in this tissue was measured as an indirect measure of the systemic distribution of test substance following inhalation exposure. At no time interval studied was test substance detected in the kidneys taken from rats exposed to the highest concentration of test substance (0.5 µg/L test substance ion). The absence of test substance in the kidney in this study suggests that the test substance present in the lung cannot be attributed to uptake from the plasma following absorption from the gastrointestinal tract, and suggests that the systemic distribution of test substance following inhalation is negligible.

Lung:
Following the exposure of rats to the lowest concentration (0.01 μg/L test substance ion) the levels of test substance in the lung were so near the limit of detection that we have disregarded these data. The amount of test substance in the lungs of rats exposed to 0.1 μg/L test substance ion reached (after 4 exposures) a steady state level of approximately 1.3 μg/g test substance wet weight lung, and remained at this level at the completion of the experiment.

The elimination half-life of test substance from the lung was determined after 1 exposure to 0.5 µg/L test substance ion and 15 exposures to 0.1 µg/L test substance ion or 0.5 µg/L test substance ion. The estimated half-life of test substance in the lung was found to be approximate to 2 days.

Following exposure to 0.5 µg/L test substance ion, the levels of test substance in the lung were maximal after 4 exposures (~5 μg/g test substance ion wet weight). The level fell within a few days to ~2 ug/g test substance ion wet weight after 10 exposures and remained there until completion of the experiment. However, between exposures 5 and 15 there was an increase in the lung wet weight. When the amount of test substance is expressed per whole lung, the peak level still occurs after 4 exposures and falls after 10 exposures, rising slightly at the completion of the experiment. In rats exposed to 0.5 µg/L test substance ion, there is a significant decrease in the amount of test substance in the lung between the 5th to the 15th exposure even when lung weight is taken into account.

Effect levels

1

Dose descriptor:

dose level:

Effect level:

>= 0.1 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other:

Remarks:

original value presented in study

2

Dose descriptor:

dose level:

Effect level:

>= 0.14 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

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:

dose level:

Effect level:

>= 0.5 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

original value presented in study

4

Dose descriptor:

dose level:

Effect level:

>= 0.69 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

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 result

The doses of the test substance were expressed in test substance 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 0.10 mg test substance ion / kg bw = 0.14 mg pure test substance / kg bw

(100/72.4) x 0.50 mg test substance ion / kg bw = 0.69 mg pure test substance / kg bw

Applicant's summary and conclusion

Conclusions:

In this 21-day repeated dose study, following exposure to test substance aerosols via inhalation, there were no significant differences between the lung test substance concentrations in male and female rats after 5 and 15 exposures at any dose level.

Executive summary:

In a repeated dose inhalation study, Sprague-Dawley (CD) were whole-body exposed to various concentrations of test substance aerosols (0,01, 0.10 and 0.50 µg/L test substance ion). The test animals were exposed 6 hours a day, 5 days a week for 3 weeks (total 15 exposures). A ultrasonic nebuliser was used to generate vapours of test substance diluted in distilled water with a size smaller than 0.7 µm. The concentration of test substance ion in each chamber was determined twice and the particle size distribution determined once during each exposure. The following objectives were addressed: 1) Whether test substance does attain a steady state concentration in rat lung following repeated exposures at 0.01, 0.1 and also 0.5 µg/L test substance ion. 2) The elimination half-life of test substance in rat lung at different exposure levels and after 1 and 15 exposures. 3) Whether there is a sex difference in lung test substance levels after repeated exposures to test substance aerosols. In the highest exposure group, pilo-erection, tachypnoea and brown nasal staining were observed in the majority of animals. The accumulation results showed that rats exposed to 0.1 µg/L test substance ion reached a steady state lung level of up to 1.3 µg/g test substance wet weight following four 6 hour exposures. The half-life of test substance in the lungs of rats exposed by inhalation is of the order of 2 days. In total 13 male rats and 1 female rat died upon exposure to 0.50 µg/L test substance ion, within the exposure window.

There were no significant differences between the lung test substance concentrations in male and female rats after 5 and 15 exposures at any dose level.