2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-06-25 to 1993-01-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

EC Number:

258-946-7

EC Name:

2-[[(4-methoxyphenyl)methylhydrazono]methyl]-1,3,3-trimethyl-3H-indolium methyl sulphate

Cas Number:

54060-92-3

Molecular formula:

C20H24N3O.CH3O4S C21H27N3O5S

IUPAC Name:

2‐{[2‐(4‐methoxyphenyl)‐2‐methylhydrazin‐1‐ylidene]methyl}‐1,3,3‐trimethyl‐3H‐indol‐1‐ium methyl sulfate

Details on test material:

- Name of test material (as cited in study report): ASTRAZON Goldgelb GL-E 200%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: young adult rats (2-3 months)
- Weight at study initiation: 165 - 205 g
- Housing: individually in macrolon type II cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical inhalation chamber, animals held in nose/head only restrainer
- Exposure chamber volume: 20 L
- Method of holding animals in test chamber: rat exposure restrainer
- Source and rate of air: clean dried compressed air, flow rate 15 L/min
- Method of conditioning air: by a downstream VIA-compressed air dryer
- System of generating particulates/aerosols: Wright-Dust_Feeder Type II (BGI Inc, Waltham, USA) and by an Exactomat 4200 (TSE, Bad Homburg, Germany)
- Method of particle size determination: by using a Berner-cascade-impactor
- Treatment of exhaust air: cleaning of the exhausted air by using cottonwool aerosol filter
- Temperature, humidity, pressure in air chamber: every 10 min by using a Leybold-Heraeus system; temperature: ca 23 °C, relative humidity (group 1 animals): 22% and relative humidity (group 2-6): < 5%.

TEST ATMOSPHERE
- Brief description of analytical method used:
At least three air samples were taken from the inhalation chamber during the exposure. The flow rate of the air was 4 L/min. To analyse the test substance concentration (mg/m³ air) Sartorius teflon filter were used located in the breathing zone of the animals. The filter were gravimetrical analysed. After this analysis the filter were eluted with acetone nitrile and the elution was further analysed by using a spectral photometer.

- Samples taken from breathing zone: yes

OTHER:
- In the test atmosphere only the dye (test item) was quantitatively present without the supporting substances sodium sulphate and further additives. This was based on separation of the dye and the supporting substances due to the smaller particle size of the dye in comparison to the coarse particle size of the supporting substances.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0, 9, 51, 75, 167 and 388 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of clinical observations: at the day of exposition several times, afterwards twice a day
- Frequency of weighing: before exposition, on day 3 and 7, afterwards once per week
- Necropsy of survivors performed: yes
- Other examinations performed:
rectal temperataur: immediately after exposure the rectal temperature was measured

Statistics:

For the graphical presentation the standard deviation was displayed in relationship to the air control value. Statistical significant changes (versus air control) were identified with * (p<0.05) and ** (p<0.01). The clinical findings were statistically evaluated with a Pairwise Fishers Test. Body weights were statistically evaluated by variance analysis (ANOVA).

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

76 mg/m³ air

Based on:

test mat.

95% CL:

55.7 - 106.2

Exp. duration:

4 h

Mortality:

All males and females of the two top concentration groups died. At the concentration of 75 mg/m³ air 2 females and 2 males died. At 51 mg/m³ air one animal of each sex died. In the lowest concentration group (9 mg/m³) no mortality occurred.

Clinical signs:

other: No adverse effects were noted in the 9 mg and 51 mg/m³ air concentration groups. The animals from the remaining groups showed the following signs of toxicity: abnormal breathing (respiratory sounds, dyspnoea), serous secretion from nostrils, loss of weigh

Body weight:

No effects seen in the lowest concentration group. At 51 mg/m³ and higher the body weight was transiently reduced in the first post-treatment days in comparison to the control group.

Gross pathology:

In all surviving animals no effects were noted after the 14 days observation period. Animals, which died during the observation period showed the following gross pathology symptoms: hepatization of the lung, lung oedema, hydrothorax, reddened nasal mucosa, yellowish, foamy trachea content, cornea opacity, test substance related skin coloration, liver with lobular structure, spleen, liver and kidney pale, reddened gastrointestinal tract, partially bloated.

Other findings:

Rectal temperature:
A toxicological relevant reduction of the rectal temperature (hypothermie) was seen at 51 mg/m³ and higher concentrations. Males showed already a mild hypothermie in the lowest concentration group.

Any other information on results incl. tables

Table 1: acute inhalation toxicity: Summary of results
Group	Concentration (mg/m³ air), (gravimetrical)		Toxicological results*		Duration of signs		Time of death	Respirability (%)
males
1	air control		0 / 0 / 5		-		-	-
2	9		0 / 0 / 5		-		-	83
3	51		1 / 5 / 5		4h - 3d		0d	47
4	75		2 / 5 / 5		4h - 7d		1d - 2d	47
5	167		5 / 5 / 5		0d		0d - 1d	77
6	388		5 / 5 / 5		< 4h		< 5h	33
females
1	air control		0 / 0 / 5		-		-	-
2	9		0 / 0 / 5		-		-	83
3	51		1 / 5 / 5		4h - 7d		4d	47
4	75		2 / 5 / 5		4h - 5d		1d	47
5	167		5 / 5 / 5		4h - 1d		4h - 2d	77
6	388		5 / 5 / 5		< 4h		< 4h	33

*= 1st number: number of dead animals, 2nd number: number of animals with clinical symptoms,

3rd number: number of animals

Table 2: Analysis of particle distributions, target concentration 8.5 mg/m³ air
N	Impactor stage (µm)		Cut-off diameter (µm)		Mass/stage (µm)	rel. Mass (%)	Cumul. Mass (%)
1	0.016 - 0.031		0.016		0.00	0.00	0.00
2	0.031-0.063		0.031		0.00	0.00	0.00
3	0.063-0.125		0.063		0.00	0.00	0.00
4	0.125-0.250		0.125		0.00	0.00	0.00
5	0.250-0.500		0.250		0.10	4.35	0.00
6	0.500-1.00		0.500		0.20	8.70	4.35
7	1.00-2.00		1.00		0.80	34.78	13.04
8	2.00-4.00		2.00		1.10	47.83	47.83
9	4.00-8.00		4.00		0.10	4.35	95.65
10	8.00-16.0		8.00		0.00	0.00	100
11	16.0-30.0		16.00		0.00	0.00	100

Mass Median Aerodynamic Diameter (MMAD): 1.69 µm

Geometric standard deviation: 1.83

Respirability (% < 3 µm):

a) mass related: 83 % (measured)

b) number related: 100 % (extrapolated)

Table 3: Analysis of particle distributions, target concentration 51.1 mg/m³ air
N	Impactor stage (µm)		Cut-off diameter (µm)		Mass/stage (µm)	rel. Mass (%)	Cumul. Mass (%)
1	0.016 - 0.031		0.016		0.00	0.00	0.00
2	0.031-0.063		0.031		0.00	0.00	0.00
3	0.063-0.125		0.063		0.00	0.00	0.00
4	0.125-0.250		0.125		0.00	0.00	0.00
5	0.250-0.500		0.250		0.00	0.00	0.00
6	0.500-1.00		0.500		0.30	6.67	0.00
7	1.00-2.00		1.00		0.70	15.56	6.67
8	2.00-4.00		2.00		2.20	48.89	22.22
9	4.00-8.00		4.00		0.90	20.00	71.11
10	8.00-16.0		8.00		0.20	4.44	91.11
11	16.0-30.0		16.00		0.20	4.44	95.56

Mass Median Aerodynamic Diameter (MMAD): 3.21 µm

Geometric standard deviation: 2.24

Respirability (% < 3 µm):

a) mass related: 47 % (measured)

b) number related: 100 % (extrapolated)

Table 4: Analysis of particle distributions, target concentration 75 mg/m³ air
N	Impactor stage (µm)		Cut-off diameter (µm)		Mass/stage (µm)	rel. Mass (%)	Cumul. Mass (%)
1	0.016 - 0.031		0.016		0.00	0.00	0.00
2	0.031-0.063		0.031		0.00	0.00	0.00
3	0.063-0.125		0.063		0.00	0.00	0.00
4	0.125-0.250		0.125		0.00	0.00	0.00
5	0.250-0.500		0.250		0.00	0.00	0.00
6	0.500-1.00		0.500		0.20	4.17	0.00
7	1.00-2.00		1.00		1.00	20.83	4.17
8	2.00-4.00		2.00		2.30	47.92	25.00
9	4.00-8.00		4.00		1.00	20.83	72.92
10	8.00-16.0		8.00		0.10	2.08	93.75
11	16.0-30.0		16.00		0.20	4.17	95.83

Mass Median Aerodynamic Diameter (MMAD): 3.20 µm

Geometric standard deviation: 2.12

Respirability (% < 3 µm):

a) mass related: 47 % (measured)

b) number related: 99 % (extrapolated)

Table 5: Analysis of particle distributions, target concentration 167 mg/m³ air
N	Impactor stage (µm)		Cut-off diameter (µm)		Mass/stage (µm)	rel. Mass (%)	Cumul. Mass (%)
1	0.016 - 0.031		0.016		0.00	0.00	0.00
2	0.031-0.063		0.031		0.00	0.00	0.00
3	0.063-0.125		0.063		0.00	0.00	0.00
4	0.125-0.250		0.125		0.00	0.00	0.00
5	0.250-0.500		0.250		0.10	2.63	0.00
6	0.500-1.00		0.500		0.20	5.26	2.63
7	1.00-2.00		1.00		0.90	23.68	7.89
8	2.00-4.00		2.00		2.40	63.16	31.58
9	4.00-8.00		4.00		0.20	5.26	94.74
10	8.00-16.0		8.00		0.00	0.00	100
11	16.0-30.0		16.00		0.00	0.00	100

Mass Median Aerodynamic Diameter (MMAD): 1.96 µm

Geometric standard deviation: 1.81

Respirability (% < 3 µm):

a) mass related: 77% (measured)

b) number related: 100 % (extrapolated)

Table 6: Analysis of particle distributions, target concentration 388 mg/m³ air
N	Impactor stage (µm)		Cut-off diameter (µm)		Mass/stage (µm)	rel. Mass (%)	Cumul. Mass (%)
1	0.016 - 0.031		0.016		0.00	0.00	0.00
2	0.031-0.063		0.031		0.00	0.00	0.00
3	0.063-0.125		0.063		0.00	0.00	0.00
4	0.125-0.250		0.125		0.00	0.00	0.00
5	0.250-0.500		0.250		0.00	0.00	0.00
6	0.500-1.00		0.500		0.10	2.13	0.00
7	1.00-2.00		1.00		0.50	10.64	2.13
8	2.00-4.00		2.00		2.20	46.81	12.77
9	4.00-8.00		4.00		1.40	29.79	59.57
10	8.00-16.0		8.00		0.10	2.13	89.36
11	16.0-30.0		16.00		0.40	8.51	91.49

Mass Median Aerodynamic Diameter (MMAD): 4.18 µm

Geometric standard deviation: 2.11

Respirability (% < 3 µm):

a) mass related: 33% (measured)

b) number related: 97 % (extrapolated)

Applicant's summary and conclusion

Interpretation of results:

Toxicity Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, the LC50 of the test item can be estimated to be 76 mg/m³ air.

Executive summary:

In an acute inhalation toxicity study equivalent to OECD 403, groups of young adult male and female Wistar rats (5 per sex/concentration) were exposed by the inhalation route (nose/head only) to the test item for 4 hours at mean concentrations of 0, 9, 51, 75, 167 and 388 mg/m³ air. Animals then were observed for 14 days. Mortality and distinct clinical signs were observed after exposure to the test substance at 51 mg/m³ air and higher. No mortality and no signs of toxicity occured after inhalation to 9 mg/m³. Based on the results obtained, the LC50 for both sexes is 76 mg/m³ air.