Diquat dibromide

Endpoint summary

Description of key information

- Oral: LD50 = 400 mg/kg bw, male/female, rat, comparable to OECD401, McCall 1990.

- Inhalation: LC50 = 0.226 mg/L, male/female, rat, comparable to OECD 403, Bruce 1985.

- Dermal: LD50 > 792 mg/kg bw, male/female, rat,  comparable to OECD 402, McCall 1990.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1990 to Mar 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 - 10 weeks old males and 7 - 11 weeks old females
- Weight at study initiation: 243 - 356 g males and 180 - 240 g females
- Fasting period before study: overnight for a period of 16 to 24 hours
- Housing: housed in suspended cages (37 cm length x 32 cm width x 20 cm height). The floor and the back of each cage were made of 1.2 cm square stainless steel mesh. The sides were made of solid stainless steel and the front was made of polycarbonate. A maximum of five rats were housed in each cage and the sexes were kept separately
- Diet: Parton Combined Diet, ad libitum
- Water: water via an automatic system, ad libitum
- Acclimation period: minimum of six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 50 ± 10
- Air changes (per hr): 20 - 30
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

dionised

Details on oral exposure:

The test substance was administered at a dose volume of 10 mL/kg bw

Doses:

100, 150, 200, 225 and 250 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Animals were observed for signs of systemic toxicity once within 2 hours of dosing and again between 4 and 7 hours after dosing. Subsequent observations were made once daily, or twice daily whenever there were significant signs of toxicity.
- The animals were weighed on the day before dosing (day-1), the day of dosing (day 1) and on susequent days thereafter.
- All animals were necropsied and examined microscopically.

Statistics:

The acute oral LD50 was calculated from the mortality data (which included animals that were killed in extremis) by logistic regression using nominal dose values. Confidence limits were calculated using a likelihood ratio interval.

Sex:

male

Dose descriptor:

LD50

Effect level:

214 mg/kg bw

Based on:

test mat.

95% CL:

>= 180 - <= 271

Remarks on result:

other: Original value presented in study

Sex:

female

Dose descriptor:

LD50

Effect level:

222 mg/kg bw

Based on:

test mat.

95% CL:

>= 203 - <= 241

Remarks on result:

other: Original value presented in study

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

400 mg/kg bw

Based on:

test mat.

95% CL:

>= 336 - <= 506

Remarks on result:

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

Sex:

female

Dose descriptor:

LD50

Effect level:

415 mg/kg bw

Based on:

test mat.

95% CL:

>= 379 - <= 450

Remarks on result:

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

Mortality:

Following a single oral dose of 100 mg/kg bw, no signs of toxicity were seen and none of the animals died. One male animal was found dead following a dose of 150 mg/kg bw and one male animal and one female animal were killed in extremis following a dose of 200 mg/kg bw. Of the animals dosed with 225 mg/kg bw two male animals and two female animals were either killed in extremis or found dead by day 8, surviving animals had recovered by day 12. All animals dosed with 250 mg/kg were either killed in extremis or found dead by day 5.

Clinical signs:

other: Following a dose of 100 mg/kg, there were no signs of toxicity. At 150 and 250 mg/kg, signs of toxicity were seen including decreased activity, hypothermia, piloerection, reduced splay reflex, distended abdomen, sides pinched in, ungroomed, upward curvatu

Gross pathology:

Two female animals dosed at 225 mg/kg bw had stomach and/or intestines filled with gas. One of these animals had pale kidneys and intrapelvic dilatation of the right kidney. The other animal had mottled kidneys. One male animal dosed at 250 mg/kg bw had stomach and intestines filled with gas.

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 oral LD50 for male rats: 1.868 x 214 = 400 mg registered substance /kg bw.

The acute oral LD50 for female rats: 1.868 x 222 = 415 mg registered substance /kg bw.

Table 1 Analysis of test substance

Nominal Concentration test substance (mg/mL)	Mean Analysed Concentration test substance (mg/mL)
10	10.2
15	15.0
20	20.3
22.5	23.0
25	25.2

Table 2 Cumulative mortality data

Day No	Dose (mg/kg) and Cumulative Mortality
	100	150	200	225	250
Male
2	0/5	0/5	0/5	0/5	1/5
3	0/5	0/5	0/5	1/5	1/5
4	0/5	1/5	0/5	2/5	4/5
5	0/5	1/5	0/5	2/5	5/5
6	0/5	1/5	1/5	2/5	5/5
15	0/5	1/5	1/5	2/5	5/5
Female
3	0/5	0/5	0/5	0/5	2/5
4	0/5	0/5	0/5	0/5	3/5
5	0/5	0/5	1/5	0/5	5/5
6	0/5	0/5	1/5	1/5	5/5
8	0/5	0/5	1/5	2/5	5/5
15	0/5	0/5	1/5	2/5	5/5

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute oral LD50 was 214 mg/kg bw test substance ion for male rats (95 % confidence limits, 180 - 271 mg/kg bw) and 222 mg/kg bw test substance ion for female rats (95 % confidence limits, 203-241 mg/kg bw).
This is equivalent to an acute oral LD50 of 400 mg/kg bw test substance salt for male rats (95 % confidence limits, 336 - 506 mg/kg bw) and 415 mg/kg bw test substance salt for female rats (95 % confidence limits, 379 - 450 mg/kg bw) of the registered substance.

Executive summary:

In this GLP compliant acute oral toxicity study, comparable to OECD guideline 401, groups of five male and five female Wistar rats each received a single oral dose of test substance technical (purity 21.1% w/w test substance ion) in deionised water at levels of 100, 150, 200, 225 or 250 mg test substance ion/kg bw and were observed up to 15 days. The animals were assessed daily for any signs of toxicity and their body weights were recorded at intervals.

Following a single oral dose of 100 mg/kg bw, no signs of toxicity were seen and none of the animals died. One male animal dosed at 150 mg/kg bw and one male and one female animal dosed at 200 mg/kg were either found dead or killed in extremis. Surviving animals at these two dose-levels showed signs of slight to moderate toxicity which persisted until day 7. Signs of extreme toxicity were seen in all animals dosed with 225 or 250 mg/kg bw. Two male and two female animals dosed with 225 mg/kg bw were either killed in extremis or found dead by day 8, although the surviving animals recovered by day 12. All animals dosed with 250 mg/kg bw were either found dead or killed in extremis by day 5.

The acute oral LD50 was 214 mg/kg bw test substance ion for male rats (95 % confidence limits, 180 - 271 mg/kg bw) and 222 mg/kg bw test substance ion for female rats (95 % confidence limits, 203-241 mg/kg bw). This is equivalent to an acute oral LD50 of 400 mg/kg bw test substance salt for male rats (95 % confidence limits, 336 - 506 mg/kg bw) and 415 mg/kg bw test substance salt for female rats (95 % confidence limits, 379 - 450 mg/kg bw) of the registered substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

400 mg/kg bw

Quality of whole database:

GLP compliant study, comparable to OECD 401.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

1981

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

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

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

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.

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

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

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

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

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

0.226 mg/L air

Physical form:

inhalation: aerosol

Quality of whole database:

GLP compliant study, comparable to OECD 403.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

1987

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

1998

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

1992

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9.5 weeks for males, 8.5 weeks for females
- Weight at study initiation: 295 - 337 g for males, 196 - 228 g for females
- Housing: Individually in suspended stainless steel and polycarbonate cages (each cage divided into 2 separate compartments)
- Diet: Porton Combined Diet, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: At least 6 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): 50 ± 10
- Air changes (per hr): 20 - 30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE dates: From Feb 1990 To: Feb 1990

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

16 - 32 hours before application, the hair was removed using veterinary clippers, from an area approximately 10 cm x 5 cm on the dorso-lumbar region of each rat. The undiluted test substance was applied to the shorn backs at a nominal rate of 2 mL/kg bw, under occlusive dressings for a period of 24 hours. At the end of the 24 hour application period, the dressings were removed and any residual test substance was removed from the skin using cotton wool swabs and clean warm water and the skin dried with tissue paper.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

EXPERIMENTAL DESIGN
A single administration was performed by the dermal route and was followed by a 15-day observation period.

CLINICAL OBSERVATIONS
The animals were examined for any signs of systemic toxicity once between 1 and 4 hours after application and then once daily for systemic toxicity and skin irritation up to day 15.

MEASUREMENT OF BODY WEIGHT
Body weights were recorded immediately before application of the test substance (day 1) and on days 3, 5, 8 and 15.

POST MORTEM INVESTIGATIONS
At the end of the scheduled observation period, all animals were necropsied and examined macroscopically.

Statistics:

No statistical analysis was used (limit test, no mortalities).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 792 mg/kg bw

Based on:

test mat.

Mortality:

There were no mortalities

Clinical signs:

other: There were no significant signs of toxicity. Black or brown staining was seen on the application sites of all but one rat, which persisted for up to 4 days in male rats and up to 15 days in females.

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

SKIN IRRITATION
Signs of moderate skin irritation (including erythema, desquamation and scabbing) were observed in all animals, persisting for the majority of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal LD50 of test substance to males and females was greater than 2000 mg/kg bw, equivalent to 792 mg/kg bw test substance salt.

Executive summary:

In an acute dermal toxicity study, comparable to OECD TG 402 and in compliance with GLP, 5 male and 5 female young adult, Alpk:APfSD rats were each given a single 24 hour dermal application of 2000 mg/kg bw of test material (equivalent to 21.2 % w/w of test substance cation). The test material was applied without further dilution to the shorn backs of the rats and held in place under an occlusive dressing. At the end of the 24 hour application period, the dressings were removed and any residual test substance was removed from the skin using cotton wool swabs and clean warm water and the skin dried with tissue paper. The animals were observed daily, for 15 days, for any signs of toxicity or dermal irritation and their body weights were recorded at intervals. At the end of the study, the animals were killed and given a gross examination post mortem. There were no mortalities and no significant signs of toxicity were seen. Signs of moderate skin irritation were seen. The body weight of the animals was within the range commonly recorded for this age and strain. No macroscopic abnormalities were detected at necropsy. The acute dermal LD50 of test substance to males and females was greater than 2000 mg/kg bw, equivalent to greater than 792 mg/kg bw test substance (salt form).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

> 792 mg/kg bw

Quality of whole database:

GLP compliant study, comparable to OECD 402.

Additional information

Acute toxicity: oral

In this GLP compliant acute oral toxicity study, comparable to OECD guideline 401, groups of five male and five female Wistar rats each received a single oral dose of test substance technical (purity 21.1% w/w test substance ion) in deionised water at levels of 100, 150, 200, 225 or 250 mg test substance ion/kg bw and were observed up to 15 days. The animals were assessed daily for any signs of toxicity and their body weights were recorded at intervals.

Following a single oral dose of 100 mg/kg bw, no signs of toxicity were seen and none of the animals died. One male animal dosed at 150 mg/kg bw and one male and one female animal dosed at 200 mg/kg were either found dead or killed in extremis. Surviving animals at these two dose-levels showed signs of slight to moderate toxicity which persisted until day 7. Signs of extreme toxicity were seen in all animals dosed with 225 or 250 mg/kg bw. In the group of animals dosed with 225 mg/kg bw, two males and two females were either killed in extremis or found dead by day 8, and the surviving animals recovered by day 12. All animals dosed with 250 mg/kg bw were either found dead or killed in extremis by day 5.

The acute oral LD50 was 214 mg/kg bw test substance ion for male rats (95 % confidence limits, 180 - 271 mg/kg bw) and 222 mg/kg bw test substance ion for female rats (95 % confidence limits, 203-241 mg/kg bw). This is equivalent to an acute oral LD50 of 400 mg/kg bw test substance salt for male rats (95 % confidence limits, 336 - 506 mg/kg bw) and 415 mg/kg bw test substance salt for female rats (95 % confidence limits, 379 - 450 mg/kg bw).

 

Acute toxicity: inhalation

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 technical (purity 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 surviving 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 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 sexes 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, corresponding to an acute inhalation LC50 for the test substance (salt form) of 0.226 mg/L for males, 0.247 mg/L for females and combined 0.234 mg/L for both sexes.

 

Acute toxicity: dermal

In an acute dermal toxicity study, comparable to OECD TG 402 and in compliance with GLP, 5 male and 5 female young adult, Alpk:APfSD rats were each given a single 24 hour dermal application of 2000 mg/kg bw of test material (equivalent to 21.2 % w/w of test substance cation). The test material was applied without further dilution to the shorn backs of the rats and held in place under an occlusive dressing. At the end of the 24 hour application period, the dressings were removed and any residual test substance was removed from the skin using cotton wool swabs and clean warm water and the skin dried with tissue paper. The animals were observed daily, for 15 days, for any signs of toxicity or dermal irritation and their body weights were recorded at intervals. At the end of the study, the animals were killed and given a gross examination post mortem. There were no mortalities and no significant signs of toxicity were seen. Signs of moderate skin irritation were seen. The body weight of the animals was within the range commonly recorded for this age and strain. No macroscopic abnormalities were detected at necropsy. The acute dermal LD50 of test substance to males and females was greater than 2000 mg/kg bw, equivalent to greater than 792 mg/kg bw test substance (salt form).

Justification for classification or non-classification

Based on the available information, the test substance is classified for Acute oral toxicity, Category 4, H302: Harmful if swallowed and Acute inhalation toxicity, Category 2, H330: Fatal if inhaled, in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Considering the very low dermal absorption of the test substance (<1%) as well as no signs of systemic toxicity were observed in the acute dermal toxicity study using a technical concentrate of the test substance up to 2000 mg/kg bw, which is  equivalent to 792 mg pure test substance/kg bw, the substance is not classified for acute dermal toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.