3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Endpoint summary

Description of key information

Acute oral toxicity: Under the conditions of the study, the LD50 of the test material was found to be 449 mg/kg bw for males and females combined (95 % confidence limit 322 to 626 mg/kg bw). The LD50 for males was 410 mg/kg bw (95 % CL 265 to 636 mg/kg bw) and for females was 453 mg/kg bw (95 % CL 266 to 771 mg/kg bw).

Acute inhalation toxicity: Under the conditions of this study, the 4 h LC50 value via the inhalation route was established to be within the range of 0.05 to 0.5 mg/L.

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:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

no

Remarks:

Study pre-dates GLP

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Albino (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Body weight prior to test material application: Males: group averages were 162 g for all groups; females: group averages were 147 g for 2 groups and 162 g for the two highest dose groups.
- Fasting period before study: Animals were deprived of food and water for 16 hours prior to test material application.
- Housing: The rats were housed in MAKROLON type III (max. 5 rats) cages consisting of a wire grid with vermiculite beneath.
- Diet: Standardised pellets for rat and mice ad libitum
- Water: ad libitum
- Acclimation period: Minimum of 3 days and maximum of 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Humidity: 55 ±1 0 % (relative)
- Photoperiod: 12 hour light/dark cycle. Light hours were from 07:00 to 19:00

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Vehicle:

other: Bi-distilled water, 0.5 % CMC-preparation olive oil (DAB7), polyethylene glycol 400

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 10, 31.6, 100 and 316 mg/mL

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: Doses were prepared immediately before application. Emulsions and suspensions were stirred with a magnetic stirrer over the whole application period.

Doses:

100, 316, 1000 and 3160 mg/kg

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations were performed <15, 15 and 30 minutes and 1, 2, 4, 5 and 24 h after application and afterwards once daily for 14 days after application. Determination of body weight was carried out immediately before dosing as well as 7 and 14 days after application. Single weighing and the body weight related to classes of the WL24-system.
- Necropsy of survivors performed: yes

Statistics:

Probit analysis following Finney was carried out (both separately and for males and females combined).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

449 mg/kg bw

Based on:

test mat.

95% CL:

>= 322 - <= 626

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

410 mg/kg bw

Based on:

test mat.

95% CL:

>= 265 - <= 636

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

453 mg/kg bw

Based on:

test mat.

95% CL:

>= 266 - <= 771

Mortality:

No mortalities were observed for the animals dosed at 100 mg/kg. At 316 mg/kg 2 females and 1 male died. In the 1000 mg/kg dose group 5 males and 4 females died and in the high dose group all rats died.

Clinical signs:

Clinical signs observed were apathy, diminished response to external stimulus, narrow eyelids, bristled fur, soft stools and reduced quantity of stools.

Body weight:

All surviving rats gained weight over the observation period, though at the 7 day evaluation 1 female in each of the 316 and 1000 mg/kg dose groups were considered to be highly emaciated.

Gross pathology:

The following findings were observed for rats that spontaneously died during the observation period. One rat was found with a lentil sized bleaching in the left side of the cardiac septum, 1 rat was observed with dark red focal region in the left pulmonary lobe, 1 rat had developed atelectasis across the whole lung, 2 rats had developed atelectasis in the apex of the lung and the whole gastro-intestinal tract was coloured red-violet, 3 rats had developed dark brown livers with light brown sprinklings, liver coloured yellowish, 3 rats were found to have red colourisation of all the internal tissues systemically, 1 rat was observed to be grey-red colourised and lastly 3 rats had one or double sided abdominal cryptorchism.
There were no findings from rats sacrificed at the end of the test.

Table 1: Summary of Mortalities

Dose rate (mg/kg)	Application volume (mL/kg)	Concentration (mg/mL)	n/5 Cumulative number of dead rats (timeframe)
			1 h		24 h		48 h		7 d		14 d
			M	F	M	F	M	F	M	F	M	F
100	10.0	10	0	0	0	0	0	0	0	0	0	0
316	10.0	31.6	0	0	0	1	1	1	1	1	1	2
1000	10.0	100	0	0	1	1	3	2	5	3	5	4
3160	10.0	316	0	0	3	3	3	3	5	5	5	5

Table 2: Summary of Body Weight

Dose rate (mg/kg)	Gender	Body weight of rats alive (g, geometric mean)			Mean factor for increase of body weight of surviving rats
		Pre appl.	7 d post appl.	14 d post appl.	n	0 - 7 d	n	7 - 14 d	n	0 - 14 d
100	M	162	196	237	5	1.21	5	1.21	5	1.47
	F	147	178	196	5	1.21	5	1.10	5	1.33
316	M	162	196	237	4	1.21	4	1.21	4	1.47
	F	147	162*	196	4	1.10	3	1.21	3	1.33
1000	M	162	—	—	—	—	—	—	—	—
	F	162	162*	215	2	1.00	1	1.33	1	1.33
3160	M	162	—	—	—	—	—	—	—	—
	F	162	—	—	—	—	—	—	—	—

*Rat highly emaciated

Interpretation of results:

other: Classified as Category 4 in accordance with EU criteria

Conclusions:

Under the conditions of the study, the LD50 of the test material was found to be 449 mg/kg bw for males and females combined (95 % confidence limit 322 to 626 mg/kg bw). The LD50 for males was 410 mg/kg bw (95 % CL 265 to 636 mg/kg bw) and for females was 453 mg/kg bw (95 % CL 266 to 771 mg/kg bw).

Executive summary:

A study was conducted to assess the acute oral toxicity potential of the test material in the rat using methodology equivalent to the standardised guideline OECD 401.

Groups of 5 female and 5 male rats were exposed the test material by gavage at dose levels of 100, 316, 1000 and 3160 mg/kg using a vehicle of bi-distilled water, 0.5 % CMC-preparation olive oil (DAB7) and polyethylene glycol 400. The animals were observed for a 14 day period for mortality and clinical signs. Necropsies were performed for animals that died spontaneously and for animals that were euthanised at the end of the study.

No mortalities were observed for the animals dosed at 100 mg/kg. At 316 mg/kg 2 females and 1 male died. In the 1000 mg/kg dose group 5 males and 4 females died and in the high dose group all rats died. Clinical signs observed were apathy, diminished response to external stimulus, narrow eyelids, bristled fur, soft stools and reduced quantity of stools. All surviving rats gained weight over the observation period, though at the 7 day evaluation 1 female in each of the 316 and 1000 mg/kg dose groups were considered to be highly emaciated.

The following findings were observed for rats that spontaneously died during the observation period. One rat was found with a lentil sized bleaching in the left side of the cardiac septum, 1 rat was observed with dark red focal region in the left pulmonary lobe, 1 rat had developed atelectasis across the whole lung, 2 rats had developed atelectasis in the apex of the lung and the whole gastro-intestinal tract was coloured red-violet, 3 rats had developed dark brown livers with light brown sprinklings, liver coloured yellowish, 3 rats were found to have red colourisation of all the internal tissues systemically, 1 rat was observed to be grey-red colourised and lastly 3 rats had one or double sided abdominal cryptorchism. There were no findings from rats sacrificed at the end of the test.

Under the conditions of the study, the LD50 of the test material was found to be 449 mg/kg bw for males and females combined (95 % confidence limit 322 to 626 mg/kg bw). The LD50 for males was 410 mg/kg bw (95 % CL 265 to 636 mg/kg bw) and for females was 453 mg/kg bw (95 % CL 266 to 771 mg/kg bw).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

410 mg/kg bw

Quality of whole database:

There is one study available that was carried out using methodology equivalent to a standardised guideline. The study pre-dates the inception of GLP however the study contains the relevant information needed for risk assessment. The quality of the database is therefore considered to be acceptable.

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:

12 August 2016 to 05 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008 including most recent amendments

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nousan, Notification No 8147

Version / remarks:

2000 including the most recent partial revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Outbred, SPF-Quality
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult animals were selected (approximately 9 to 10 weeks old)
- Weight at study initiation: Males: 183 to 358 g; females: 174 to 210 g. Body weight variation did not exceed ± 20 % of the sex mean.
- Fasting period before study: No
- Housing: Group housing of five animals per sex per cage (height 18 cm) containing sterilised sawdust as bedding material and paper as cage-enrichment. After exposure, the surviving animals were returned to their cages which were placed in a fume cupboard for a short time period to allow test material remnants to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in case of bad health condition and in order to recover and to aid the clinical observations. The sheet was removed before the end of the exposure day and the surviving animals were returned to the animal room.
- Diet: Free access to pelleted rodent diet except during exposure to the test material
- Water: Free access to tap water except during exposure to the test material
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: Not reported

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.5 - <= 4 µm

Geometric standard deviation (GSD):

>= 1.5 - <= 2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The design of the exposure chamber is based on the flow past nose-only inhalation chamber. The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.
- Exposure chamber volume: Approximately 150 mL
- Method of holding animals in test chamber: The animals were placed in restraining tubes and connected to the animal ports. Prior to each exposure, both eyes of each rat were instilled with Opthosan to protect the eyes against potential irritation by the test material. Prior to exposure the animals were restrained in polycarbonate restraining tubes; these tubes were connected to the exposure chamber. Sixteen (5 mg/L), twenty-two (0.5 mg/L) or twenty (0.05 mg/L) minutes after the last animal was placed the generation of the test atmosphere was started.
- Source and rate of air: The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal. For the 1, 0.5 and 0.05 mg/L concentrations, the mean total airflows were 17, 33 and 62 L/min, respectively.
- System of generating particulates/aerosols: The test atmosphere generation was based on the method developed during trial generations. For the generation of 1 mg/L, a dust was generated by administering the test material to a stream of pressurised air using a combination of a brush feeder and micronising jet mill. The dust was passed through a series of three cyclones, allowing larger particles to settle, and diluted with pressurised air before it entered the exposure chamber. The mean total airflow was 17 L/min.
For the generation of 0.5 and 0.05 mg/L, a dust was generated by administering the test material to a stream of pressurised air using a combination of a spiral feeder and an air mover. The dust was passed through a series of four or five cyclones, allowing larger particles to settle, and diluted with pressurised air before it entered the exposure chamber. The mean total airflows were 33 L/min (for 0.5 mg/L) and 62 L/min (for 0.05 mg/L).
- Method of particle size determination: The particle size distribution was characterised once during the exposure period of 1 and 0.5 mg/L and twice during the 0.05 mg/L period. The samples were drawn (at 2 or 10 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fibre glass filters (TE-290-GF,Tisch Environmental, Cleves, Ohio, USA) and a fibre glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test material collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined.
- Treatment of exhaust air: From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity were measured with a humidity and temperature indicator and were recorded after the animals were placed in the experimental set-up and at 30 minute intervals after initiation of the exposure. The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The temperature of the atmosphere during the exposures was between 21.3 and 22.1 °C and relative humidity was between 20 and 42 %. These conditions were considered appropriate for this relatively short 4 hours exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method used: The nominal concentration was calculated by dividing the amount of test material used by the volume of pressurised air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.
A total of 9, 13 and 17 representative samples were taken for determination of the actual concentration during exposure at 1, 0.5 and 0.05 mg/L, respectively. Samples were drawn through a glass fibre filter (type APFC04700, Millipore, Billerica, MA, USA). The collected amount of the test material in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands). Time-weighted mean concentrations with the standard deviations were calculated.
- Samples taken from breathing zone: Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber.

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): At 1 mg/L, the MMAD was 3.6 μm (gsd 2.0). At 0.5 mg/L, the MMAD was 4.0 μm (gsd 1.9). At 0.05 mg/L, the MMAD was 2.7 μm (gsd 1.5) and 2.5 μm (gsd 1.5).

METHOD
- Rationale for the selection of the starting concentration: The study was initiated with the exposure of five animals of each sex to a target concentration of the test material of 1 mg/L. Based on the results, two groups of five males and females were exposed in a step wise fashion to target concentrations of 0.5 and 0.05 mg/L.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.05, 0.5 and 1 mg/L

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for mortality/viability were made twice daily. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe were sacrificed for humane reasons. During exposure animals were observed three times for mortality, behavioural signs of distress and effects on respiration. After exposure animals were observed for clinical signs on Day 1, one and three hours after exposure and once daily thereafter until Day 15. The clinical signs were graded according to fixed scales. Body weights were recorded on Days 1 (pre-administration), 2, 4, 8 and 15 and at death (if found dead or sacrificed after Day 1).
- Necropsy of survivors performed: Yes. The moribund animals and animals surviving to the end of the observation period were sacrificed by an intraperitoneal injection with Euthasol®. Descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract.

Statistics:

No statistical analysis was performed.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.05 - 0.5 mg/L air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

At 1 and at 0.5 mg/L, all animals were found dead or sacrificed for ethical reasons within 3 hours after start of the exposure. At 0.05 mg/L, two males were sacrificed for ethical reasons on Day 3 or day 6. No further mortality occurred.

Clinical signs:

other: At 0.5 mg/L, slow breathing was seen during the exposure. No signs were recorded during the exposure to 1 mg/L and since the animals died during exposure to 1 and 0.5 mg/L, no clinical observations were performed after exposure. At 0.05 mg/L, slow breathi

Body weight:

Reduced body weight gain and body weight loss was seen for the surviving animals exposed to 0.05 mg/L during the first week after exposure. All animals gained in weight during the second week.

Gross pathology:

At 1 mg/L, purple discolouration of the trachea and dark red discolouration of the lungs were observed. At 0.5 mg/L, foamy contents and/or purple discolouration of the trachea, dark red discolouration of the lungs, purple discolouration of the stomach, accentuated lobular pattern of the liver and reddish isolated foci of the thymus were observed. At 0.05 mg/L one of the two animals that were killed in extremis showed irregular surface of the stomach and the thymus was reduced in size. The other one showed the gastro-intestine tract distended with gas. No abnormalities were seen for the surviving animals at 0.05 mg/L.

Other findings:

- Test Atmosphere Characterisation
For the target exposure to 1 mg/L, the time-weighted mean actual concentration was 1.2 ± 0.1 mg/L. The nominal concentration (amount of test material used divided by the volume of pressurised air used) was 81 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 1.6 %.
For the target exposure to 0.5 mg/L, the time-weighted mean actual concentration was 0.5 ± 0.02 mg/L. The nominal concentration (amount of test material used divided by the volume of pressurised air used) was 17 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 29 %.
For the target exposure to 0.05 mg/L, the time-weighted mean actual concentration was 0.06 ± 0.003 mg/L. The nominal concentration (amount of test material used divided by the volume of pressurised air used) was 2.5 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 2 %.
The concentration measurements equally distributed over time showed that the material was sufficiently stable. Short drops in opacity were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculating the time-weighted mean concentration, the effects of these variations were taken into account resulting in an actual reflection of the mean exposure concentration over time.

Interpretation of results:

other: Classified as Category 2 in accordance with EU criteria

Conclusions:

Under the conditions of this study, the 4 h LC50 value via the inhalation route was established to be within the range of 0.05 to 0.5 mg/L.

Executive summary:

The potential of the test material to cause acute inhalation toxicity was investigated in accordance with the standardised guidelines OECD 403, EU Method B.2, US EPA OPPTS 870.1300 and JMAFF 12 Nousan Notification No 8147 under GLP conditions.

The test material was administered as a dust by nose-only inhalation for up to 4 hours to three groups of five male and five female Wistar rats at concentrations of 0.05, 0.5 and 1 mg/L. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

For the target exposure to 1 mg/L, the time-weighted mean actual concentration was 1.2 ± 0.1 mg/L. The nominal concentration (amount of test material used divided by the volume of pressurised air used) was 81 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 1.6 %. The duration of the exposure was 81 minutes due to the mortality observed.

For the target exposure to 0.5 mg/L, the time-weighted mean actual concentration was 0.5 ± 0.02 mg/L. The nominal concentration was 17 mg/L. The generation efficiency was 29 %. The duration of the exposure was 146 minutes due to the mortality observed.

For the target exposure to 0.05 mg/L, the time-weighted mean actual concentration was 0.06 ± 0.003 mg/L. The nominal concentration was 2.5 mg/L. The generation efficiency was 2 %. The duration of the exposure was 240 minutes.

The concentration measurements equally distributed over time showed that the material was sufficiently stable. Short drops in opacity were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculating the time-weighted mean concentration, the effects of these variations were taken into account resulting in an actual reflection of the mean exposure concentration over time.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined once or twice during the exposure period. At 1 mg/L, the MMAD was 3.6 μm (gsd 2.0). At 0.5 mg/L, the MMAD was 4.0 μm (gsd 1.9). At 0.05 mg/L, the MMAD was 2.7 μm (gsd 1.5) and 2.5 μm (gsd 1.5).

At 1 and at 0.5 mg/L, all animals were found dead or sacrificed for ethical reasons within 3 hours after start of the exposure. At 0.05 mg/L, two males were sacrificed for ethical reasons on Day 3 or day 6. No further mortality occurred.

At 0.5 mg/L, slow breathing was seen during the exposure. Since the animals died during exposure to 1 and 0.5 mg/L, no clinical observations were performed after exposure.

At 0.05 mg/L, slow breathing was seen during the exposures. After exposure lethargy, hunched posture, laboured respiration, rales, gasping, swelling of the abdomen and piloerection was seen for the animals between Days 1 and 12. General purple staining by the test material was observed throughout the observation period.

Reduced body weight gain and body weight loss was seen for the surviving animals exposed to 0.05 mg/L during the first week after exposure. All animals gained in weight during the second week.

Macroscopic post mortem examination of the animals that were found dead or sacrificed for ethical reasons during the study revealed:

At 1 mg/L, purple discolouration of the trachea and dark red discolouration of the lungs.

At 0.5 mg/L, foamy contents and/or purple discolouration of the trachea, dark red discolouration of the lungs, purple discolouration of the stomach, accentuated lobular pattern of the liver and reddish isolated foci of the thymus.

At 0.05 mg/L, one of the two animals that were killed in extremis showed irregular surface of the stomach and the thymus was reduced in size. The other one showed the gastro-intestine tract distended with gas. No abnormalities were seen for the surviving animals (0.05 mg/L).

Under the conditions of this study, the 4 h LC50 value via the inhalation route was established to be within the range of 0.05 to 0.5 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

50 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute oral toxicity

A study was conducted to assess the acute oral toxicity potential of the test material in the rat using methodology equivalent to the standardised guideline OECD 401. The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Groups of 5 female and 5 male rats were exposed the test material by gavage at dose levels of 100, 316, 1000 and 3160 mg/kg using a vehicle of bi-distilled water, 0.5 % CMC-preparation olive oil (DAB7) and polyethylene glycol 400. The animals were observed for a 14 day period for mortality and clinical signs. Necropsies were performed for animals that died spontaneously and for animals that were euthanised at the end of the study.

No mortalities were observed for the animals dosed at 100 mg/kg. At 316 mg/kg 2 females and 1 male died. In the 1000 mg/kg dose group 5 males and 4 females died and in the high dose group all rats died. Clinical signs observed were apathy, diminished response to external stimulus, narrow eyelids, bristled fur, soft stools and reduced quantity of stools. All surviving rats gained weight over the observation period, though at the 7 day evaluation 1 female in each of the 316 and 1000 mg/kg dose groups were considered to be highly emaciated.

The following findings were observed for rats that spontaneously died during the observation period. One rat was found with a lentil sized bleaching in the left side of the cardiac septum, 1 rat was observed with dark red focal region in the left pulmonary lobe, 1 rat had developed atelectasis across the whole lung, 2 rats had developed atelectasis in the apex of the lung and the whole gastro-intestinal tract was coloured red-violet, 3 rats had developed dark brown livers with light brown sprinklings, liver coloured yellowish, 3 rats were found to have red colourisation of all the internal tissues systemically, 1 rat was observed to be grey-red colourised and lastly 3 rats had one or double sided abdominal cryptorchism. There were no findings from rats sacrificed at the end of the test.

Under the conditions of the study, the LD50 of the test material was found to be 449 mg/kg bw for males and females combined (95 % confidence limit 322 to 626 mg/kg bw). The LD50 for males was 410 mg/kg bw (95 % CL 265 to 636 mg/kg bw) and for females was 453 mg/kg bw (95 % CL 266 to 771 mg/kg bw).

Acute inhalation toxicity

The potential of the test material to cause acute inhalation toxicity was investigated in accordance with the standardised guidelines OECD 403, EU Method B.2, US EPA OPPTS 870.1300 and JMAFF 12 Nousan Notification No 8147 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was administered as a dust by nose-only inhalation for up to 4 hours to three groups of five male and five female Wistar rats at concentrations of 0.05, 0.5 and 1 mg/L. Mortality and clinical signs were observed daily during the observation period and body weights were determined on Days 1, 2, 4, 8 and 15. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

For the target exposure to 1 mg/L, the time-weighted mean actual concentration was 1.2 ± 0.1 mg/L. The nominal concentration (amount of test material used divided by the volume of pressurised air used) was 81 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 1.6 %. The duration of the exposure was 81 minutes due to the mortality observed.

For the target exposure to 0.5 mg/L, the time-weighted mean actual concentration was 0.5 ± 0.02 mg/L. The nominal concentration was 17 mg/L. The generation efficiency was 29 %. The duration of the exposure was 146 minutes due to the mortality observed.

For the target exposure to 0.05 mg/L, the time-weighted mean actual concentration was 0.06 ± 0.003 mg/L. The nominal concentration was 2.5 mg/L. The generation efficiency was 2 %. The duration of the exposure was 240 minutes.

The concentration measurements equally distributed over time showed that the material was sufficiently stable. Short drops in opacity were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculating the time-weighted mean concentration, the effects of these variations were taken into account resulting in an actual reflection of the mean exposure concentration over time.

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined once or twice during the exposure period. At 1 mg/L, the MMAD was 3.6 μm (gsd 2.0). At 0.5 mg/L, the MMAD was 4.0 μm (gsd 1.9). At 0.05 mg/L, the MMAD was 2.7 μm (gsd 1.5) and 2.5 μm (gsd 1.5).

At 1 and at 0.5 mg/L, all animals were found dead or sacrificed for ethical reasons within 3 hours after start of the exposure. At 0.05 mg/L, two males were sacrificed for ethical reasons on Day 3 or day 6. No further mortality occurred.

At 0.5 mg/L, slow breathing was seen during the exposure. Since the animals died during exposure to 1 and 0.5 mg/L, no clinical observations were performed after exposure.

At 0.05 mg/L, slow breathing was seen during the exposures. After exposure lethargy, hunched posture, laboured respiration, rales, gasping, swelling of the abdomen and piloerection was seen for the animals between Days 1 and 12. General purple staining by the test material was observed throughout the observation period.

Reduced body weight gain and body weight loss was seen for the surviving animals exposed to 0.05 mg/L during the first week after exposure. All animals gained in weight during the second week.

Macroscopic post mortem examination of the animals that were found dead or sacrificed for ethical reasons during the study revealed:

At 1 mg/L, purple discolouration of the trachea and dark red discolouration of the lungs.

At 0.5 mg/L, foamy contents and/or purple discolouration of the trachea, dark red discolouration of the lungs, purple discolouration of the stomach, accentuated lobular pattern of the liver and reddish isolated foci of the thymus.

At 0.05 mg/L, one of the two animals that were killed in extremis showed irregular surface of the stomach and the thymus was reduced in size. The other one showed the gastro-intestine tract distended with gas. No abnormalities were seen for the surviving animals (0.05 mg/L).

Under the conditions of this study, the 4 h LC50 value via the inhalation route was established to be within the range of 0.05 to 0.5 mg/L.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to acute oral toxicity as Category 4 (H302; Harmful if swallowed) and with respect to acute inhalation toxicity as Category 2 (H330: Fatal if inhaled).