2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium dihydrogen phosphate

Endpoint summary

Description of key information

Acute oral toxicity: LD50 = 1600 mg/kg in rats.

Acute inhalation toxicity: no mortality up to 2.345 mg/l and 0.688 mg/l in rats.

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:

from January to May, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Not specified.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 - 14 weeks of age.
- Weight at study initiation: 155 - 191 g (males), 152 - 189 m (females).
- Housing: 5 per cage.
- Diet: pellet suspended from 16 h before to 4 h post exposure.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 0.5 °C.
- Humidity: 60 ± 5 %.
- Photoperiod: 12 hrs dark / 12 hrs light.

Route of administration:

oral: gavage

Vehicle:

water

Doses:

1000, 2000, 2500, 3100, 5000 mg/kg bw.

No. of animals per sex per dose:

5 animal/sex/dose.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- Frequency of observations: twice daily.
- Other examinations performed: clinical signs (position, sedation, diarrhoea); histopathological examination of dead animals.

Statistics:

LD50 value with p lower than 0.05 computed based on Rosiello et al., J. Tox. Environ. Health 3, 1977, 797.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 400 mg/kg bw

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 1 600 mg/kg bw

Based on:

act. ingr.

Mortality:

Mortality occurred within 2 hours from administration.

Clinical signs:

Symptoms, as reduction of general conditions, sedation and diarrhoea, were evident starting from 10 minutes up to 3 days after administration.

Gross pathology:

Stomach and intestines of dead animals dosed from 2000 to 5000 mg/kg bw could not be evaluated due to discoloration.
The other organs at dosages 1000-3100 mg / kg showed no compound-related changes.

Results in female rats

doses mg/kg	no. animals	no. dead animals	no. animals with symptoms	time of death	duration of symptoms
1000	5	0	0
2000	5	1	5	2 h	10 min to 3 d
2500	5	3	5	2 h	10 min to 3 d
3100	5	5	5	2 h	10 min to 3 d
5000	5	5	5	1h 1/2	10 min to 3 d

Results in male rats

doses mg/kg	no. animals	no. dead animals	no. animals with symptoms	time of death	duration of symptoms
1000	5	0	0
2000	5	0	5		10 min to 3 d
2500	5	2	5	2 h	10 min to 3 d
3100	5	4	5	1 h 1/2	10 min to 3 d
5000	5	5	5	1h 1/2	10 min to 3 d

Overall mortality

doses mg/kg	no. animals	mortality %
1000	10	0
2000	10	10
2500	10	50
3100	10	90
5000	10	100

Interpretation of results:

other: Category 4 based on CLP criteria

Conclusions:

Acute oral exposure of rats to the substance lead to LD50 = 2400 mg/kg bw, equivalent to ca. 1600 mg/kg bw as active ingredient. Higher toxicity in female than in male rats was noted.

Executive summary:

Method

Acute oral toxicity by gavage in both male and female rats with a 14-day observation period.

Results

LD50 of 2400 mg/kg for both male and female rats, equivalent to ca. 1600 mg/kg bw of active ingredient for the given purity of test material. Female rats showed higher sensitivity than males to the substance. Mortality occurred within 2 hours from administration. At necropsy, stomach and intestines of dead animals dosed from 2000 to 5000 mg/kg bw could not be evaluated due to discoloration; the other organs at dosages 1000 - 3100 mg / kg bw showed no compound-related effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 600 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

from March 26 to April 12, 1990

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:

1981

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 2 -3 months.
- Weight at study initiation: 180 - 210 g.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C.
- Humidity: ca. 50 %.
- Air changes: 10 per hr.
- Photoperiod: 12 h light / 12 h dark.

Route of administration:

other: inhalation of aerosol or dust

Type of inhalation exposure:

nose/head only

Vehicle:

other: water (aerosol)

Details on inhalation exposure:

CHAMBER DESCRIPTION
- exposure chamber volume: ca. 20 l
- air: compressed air at 8 - 10 bar
- method of conditioning air: removal of water, dust and oil

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION: aerosol
- exposure apparatus: inhalation chamber with pre-separator.
- aerosol generation: with nozzle and conditioned compressed air (10 l of air per minute, dispersion pressure 500 kPa). 500000 µl/m3 of air of 12 % aqueous dilution were nebulised under dynamic conditions in the pre-separator of the inhalation chamber. Container with the aqueous test sample dilution was continuously stirred using a magnetic stirrer. The solution was transferred using a peristaltic pump into the nozzle.
- control of particle size: pre-separator increases efficiency of aerosol generation and also allows deposition of larger particles.
- air exchange: 30 times per hour.
- equilibrium: reached in 6 minutes.
- treatment of exhaust air: 80 % through cotton-wool filter (aerosol filter).

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION: dust
- exposure apparatus: inhalation chamber without pre-separator.
- system of generating particulates: dust generated by 'Exactomat 4200'.
- air supply: 28 l/min.
- air discharged: 20 l/min.
- air exchange: 84 times per hour.
- equilibrium: reached in 6 minutes.
- treatment of exhaust air: absolute filter.

TEST ATMOSPHERE
- dust temperature and humidity: ca 22 °C and 20 - 30 %.
- aerosol temperature and humidity: ca 23 °C and > 90 %.
- temperature and humidity in air chamber: measured every 10 minutes.
- reference group: exposure 6 h/day for 5 days.
- test group: 4/h exposure.
- brief description of analytical method used: a humidity sensor was used in which a water-attracting polymer serves as a dielectric. As a function of the moisture content of the ambient, air penetrates into water as polymer and causes a large change in the capacitance of the capacitor. The moisture measuring cell is protected by a Teflon membrane against aerosols and dust particles. The calibration of the sensors is wet with saturated salt solutions. The calibration of the temperature sensors was carried out with a standard thermometer.

ANALITICS OF TEST ATMOSPHERE : aerosol
- nominal concentration calculated from the ratio of the sprayed into the pre-separator of the inhalation chamber test sample (mg) and the total air flow rate per inhalation chamber (m3). The analytical lower concentrations - as compared to the nominal concentrations - attributed to the deposition of large particles in the pre-separator. The determination of the actual specimen concentration in the breathing zone of rats was performed with the help of filter analyses dried on gravimetric determination. 10-20 l of air in the test atmosphere breathing region of the rats were removed with a flow of 4 l/min per analysis.
- aerosol distribution: adequate to expose all potential target structures of the respiratory tract.

ANALITICS OF TEST ATMOSPHERE: dust
- indication of a nominal concentration reading is not possible for technical reasons. Determination of the actual sample concentration in the breathing zone of rats was performed with the help of filter analyses evaluated gravimetrically.
- particles features: sampling for analysis of particle distribution was carried out in the immediate breathing zone of rats. The individual impactor stages were evaluated gravimetrically.
- particle size distribution: adequate to expose all potential target structures of the respiratory tract.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Analytic concentration of dust: 943 and 5070 mg/m3 air.
Analytic concentration of aerosol: 1075 mg/m3 air.
Nominal concentration of aerosol: 60000 mg/m3 air.

No. of animals per sex per dose:

5 animal/sex/dose.

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: daily.
- Necropsy of survivors performed: yes.
- Other symptoms considered in animal assessment: respiration, circulation, mucous membranes of eyes and respiratory tract, condition of snout skin and ears, coat condition, cleaning activities, somatomotor and behaviour patterns, central nervous system.

Statistics:

Pairwise Fisher's Test and Chi-Square-Test.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 070 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: dust

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 245 mg/m³ air (analytical)

Based on:

act. ingr.

Exp. duration:

4 h

Remarks on result:

other: dust

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 075 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: aerosol

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 688 mg/m³ air (analytical)

Based on:

act. ingr.

Exp. duration:

4 h

Remarks on result:

other: aerosol

Mortality:

Dust and aerosol exposure: no mortality up to the highest doses.

Body weight:

Dust exposure: significant difference only in group 3 (5070 mg/m3 air).
Aerosol exposure: significant difference only in group 2 (1075 mg/m3 air).

Gross pathology:

Dust and aerosol exposure: no evidence of specific organ changes.

Dust exposure

The analysis of the particle size distribution of the dust in the breathing zone of the rats did not produce adequate respirable mass fraction .

Group 1 (control): all rats tolerated the treatment clinically asymptomatic.
Group 2 and 3 (943 and 5070 mg/m³air): dyspnea, respiratory sounds, slow and difficult breathing, decreased motility, laxity, serous nasal discharge and unkempt fur.

Qualitative differences between group 2 and 3 were not evident. With regard to the duration of symptoms, however, a concentration dependency was seen. Group 3 rats showed strong respiratory tract irritation.

The relatively rapid reversibility of respiratory tract irritation, the overall low inhalability of dust and the only slightly pronounced concentration dependence lead to the conclusion that the irritation is induced by a particle deposition in the upper respiratory tract or in the nose and throat.

group	sex	no. animals	conc. mg/m^3 air	dead animals	animals with symptoms	symptom duration	% particles ≤ 3 µm
1	M	5	0	0	0	-	-
2	M	5	943	0	5	4h - 1d	4
3	M	5	5070	0	5	4h - 2d	1
1	F	5	0	0	0	-	-
2	F	5	943	0	5	4h - 2d	4
3	F	5	5070	0	5	4h - 2d	1

Aerosol exposure

The analysis of the particle size distribution of the aerosol in the breathing zone of the rats revealed a high inhalable mass fraction.

Group 1 (control): all rats tolerated the treatment clinically asymtomatic.

Group 2 (1075 mg/m³air): slow and difficult breathing, reduced motility, unkempt and ruffled fur.

group	sex	no. animals	conc. mg/m^3 air	dead animals	animals with symptoms	symptom duration	% particles ≤ 3 µm
1	M	5	0	0	0	-	-
2	M	5	1075	0	5	4h - 5d	61
1	F	5	0	0	0	-	-
2	F	5	1075	0	5	4h - 3d	61

Interpretation of results:

study cannot be used for classification

Remarks:

no mortality up to the highest tested doses, which are below the threshold fof classification within the CLP Regulation (EC 1272/2008)

Conclusions:

Overall, dust and aerosol exposure showed that, via the respiratory tract, particles had no particular acute inhalation toxicity. Indeed, no mortalities were recorded up to the highest tested doses. Thus, LD50 > 1075 and 5070 mg/m3 air for aerosol and dust respectively.
However, high concentrations of particulate material, as dust, may cause respiratory tract irritation due to physical properties of the dust, which dries the mucous membranes of the upper respiratory tract.

Executive summary:

Method

Acute toxicity by inhalation of dust particles or aerosol in male and female rats with a 14 -day observation period.

Results

No mortalilty was recorded up to the highest tested concentrations, namely 5070 mg/m³ air as dust and 1075 mg/m³ air as aerosol, i.e. 3245 and 688 mg active ingredient/m³ air . Accordingly, no LC50 could be identified.

A large fraction of aerosol particles is respirable, while upon dust exposure, irritation of the respiratory tract was seen.

Symptoms, as slow/difficult breathing and reduced motility, were reversible under test conditions, with duration depending on physical form (dust or aerosol) of the substance, concentration of the substance and sex of animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

As for the oral route, acute toxicity of the substance was tested in rats by gavage. The parameters observed at periodic intervals up to day 15 were mortality, clinical signs and gross pathology of dead animals.

The results are reported: death occurred within 2 hours from exposure, starting from 2500 mg/kg bw in males and 2000 mg/kg bw in females (equal to 1650 and 1320 mg active ingredient/kg bw).

Stomach and intestines of dead animals dosed from 2000 to 5000 mg/kg bw could not be evaluated due to discoloration, while other organs at dosages 1000 -3100 mg / kg showed no compound-related effects.

Symptoms, in terms of reduction of general conditions, diarrhoea, sedation, ... were already seen at the lowest tested dose of 1000 mg/kg bw.

Acute toxicity via inhalation was tested in rats with a 4 -hour exposure followed by a 14 -day observation period. Test substance was administered as dust or aerosol. As for dust, the fraction of respirable particles (≤ 3 µm) did not exceed 4 %, while it was 61 % for aerosol.

The parameters observed at periodic intervals up to day 15 were: mortality; body weights; clinical signs, focusing on general behaviour, respiration, eye, nose, motility and other possible signs; macroscopic findings after necropsy of all animals at day 15.

No mortalilty was seen up to the highest tested concentrations, corresponding to 3245 mg active ingredient / m³ air as dust and 688 mg active ingredient / m³ air as aerosol. Accordingly, no LC50 could be identified.

Main findings were irritation of the respiratory tract, mainly for dust, and change in body weight. Duration of symptoms, reversible in all cases, showed dependence on physical form (aerosol or dust) and concentration of the substance and sex of exposed animals.

No organ abnormalities at post mortem examination were seen upon inhalation.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), the threshold of classification for acute oral toxicity is 2000 mg/kg bw. Based on the content of active ingredient, the substance has a LD50 of ca. 1600 mg/kg bw in rats. Accordingly, it is classified as category 4 for acute oral toxicity.

According to the CLP Regulation (EC 1272/2008), the threshold of classification for acute toxicity upon inhalation of dust or aerosol is 5 mg/l. As for this route of exposure, none mortality was detected up to the highest test dose of 3.245 and 0.688 mg/l, for inhalation of dust and aerosol respectively. Accordingly, no conclusion on classification for acute toxicity via inhalation could be drawn.