2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Endpoint summary

Description of key information

In an acute oral toxicity study (Grundler, 1981) conducted with the target substance (18.3%) in a mixture, the combined oral LD50 was determined to be 3.680 mg/kg bw. By extrapolating the concentration of 18.3% of the target substance within the mixture to 100% the calculated combined LD50 value would be 675 mg/kg bw.

In a second acute oral toxicity study (Hackenberg, 1977) conducted with the target substance (20%) in a mixture, the combined oral LD50 was determined to 2.060 mg/kg bw. By extrapolating the concentration of 20% of the target substance within the mixture to 100% the calculated LD50 value would be 412 mg/kg bw for both sexes.

Supporting information is provided by the study by Katz, 1970 in a read-across approach to get information on the toxicity of the chromophore itself. In this study, the acute oral LD50 in rats was determined to be 3.700 mg/kg bw in males and 4.300 mg/kg bw in females. Details on the read-across rational are provided in section 13.

No data regarding the target substance was available for acute toxicity via the inhalation route. Furthermore, as the substance is only marketed as an aqueous solution containing significant concentrations of free acetic acid as stabiliser, this route of exposure is not relevant and animal testing is not considered to be in accordance with animal welfare regulations. Thus, available data from the chloride salt of the target substance was used in a read-across approach solely to get information on the toxicity of the chromophore itself. Based on the results obtained from an in vivo acute inhalation study, the combined LC0 value of the source substance can be considered to be 1.0408 mg/L.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981-10-13 to 1981-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: ca. 12 weeks old
- Housing: 5 animals were kept in per cage (V-II-A-steel cages)
- Fasting period before study: 16 hours before treatment animals received no diet
- Diet (e.g. ad libitum): SSNIFF R from SSNIFF Versuchstierdiaeten, Soest, Germany; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 45 -75
- Air changes (per hr): not identified
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

2150, 3160, 4640 and 6810 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: <15 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 5 hours after application. Afterwards once per day the clinical signs were recorded and twice per day mortality was checked.
- Frequency of weighing: before application, and on days 3, 7 and 13 after application
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs

Statistics:

Probit analysis [Finney D.J., 1971; Cambridge University Press Vol. 3]

Sex:

female

Dose descriptor:

LD50

Effect level:

2 790 mg/kg bw

Based on:

test mat.

Sex:

male

Dose descriptor:

LD50

Effect level:

4 640 mg/kg bw

Based on:

test mat.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

3 680 mg/kg bw

Based on:

test mat.

Mortality:

Please see table 1 in the box "Any other information on results incl. tables".

Clinical signs:

Clincal signs were observed in all dose groups. Please see table 2 in the box "Any other information on results incl. tables".

Body weight:

No effects on body weight development.

Gross pathology:

Sacrificed animals without any findings in organs.
Moridund or dead animals (4640/6810 mg/kg bw): Hyperemia, blue colouration of the instestinal mucosa, partly atonic, dilated, several organs, muscles and fat tissue coloured blue.

Table 1: Mortality
Dose (mg/kg bw)		6810	4640	3160	2150
Number of animals, male		5	5	5	5
Dead animals after
1 hour		0	0	0	0
1 day		2	0	0	0
2 days		4	2	0	0
7 days		4	3	0	0
14 days		4	3	0	0
Number of animals, female		5	5	5	5
Dead animals after
1 hour		0	0	0	0
1 day		4	4	0	1
2 days		5	4	0	1
7 days		5	5	2	1
14 days		5	5	3	1

Table 2: Signs of toxicity
Dose (mg/kg bw)		6810	4640	3160	2150
Clinical signs, male
Dyspnoe		15M-6D	15M-7D	30M-3D	30M-5H
Abnormal breething				7D
Apathy		15M-6D	15M-2D	30M-3D	30M-5H
Increased spontaneous activity			3D-7D
Prone Position		1H
Ataxia		15M-6D	15M-7D	30M-3D	30M-5H
Tremble		30M-2D	1H-2D	1H
Spastic gait		2D-3D	2D-7D
Blue feces		5H	5H	5H
Blue urine		1H-3D	4H-3D	5H-3D	1D-2D
Piloerection		4H-6D	2H-7D	2H-3D	4H-2D
Diarrhoea		5H	5H	5H
Salivation			4H-5H
Ptosis		1H-2D	1H-2D
Cachexia		3D
Poor general condition		15M-6D	15M-7D	30M-3D	30M-5H
Clinical signs, female
Dyspnoe		15M-1D	15M-3D	30M-10D	30M-1D
Apathy		15M-1D	15M-3D	30M-10D	30M-1D
Aggressive behaviour				3D-7D
Prone Position		1H	1H-1D
Ataxia		15M-1D	15M-3D	30M-10D	30M-5H
Tremble		30M-1D	1H-2D	1H-8D	1H
Spastic gait			2D-3D	2D-8D
Blue feces		5H	5H	5H
Blue urine		1H-1D	4H-3D	5H-3D	1D-2D
Piloerection		4H-1D	4H-3D	2H-10D	2H-3D
Exsiccoseis			1D-2D	8D
Salivation			4H-5H
Ptosis			1H-1D
Cachexia			3D	3D
Diarrhoea		5H	5H	5H
Poor general condition		15M-1D	15M-3D	30M-10D	30M-1D

D=day

H=hour

M=Minute

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute oral toxicity study, the combined LD50 of the test item was determined to be 3680 mg/kg bw in female and male Sprague-Dawley rats. By extrapolating the concentration of 18.3% of the target substance within the mixture to 100% the calculated LD50 value would be 675 mg/kg bw and classification as Acute Tox 4, H302 is warranted in accordance with the CLP regulation.

Executive summary:

In an acute oral toxicity study, the combined LD50 of the test item was determined to be 3680 mg/kg bw in female and male Sprague-Dawley rats. By extrapolating the concentration of 18.3% of the target substance within the mixture to 100% the calculated LD50 value would be 675 mg/kg bw and classification as Acute Tox 4, H302 is warranted in accordance with the CLP regulation.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977-09-29

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:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Mean: males 147 g, females 133 g
- Fasting period before study: ca. 16 hours before application
- Housing: cages: MAKROLON type III cages (max. 5 rats),
- Diet (e.g. ad libitum): ad libitum, standard pellet diet for rats and mice
- Water (e.g. ad libitum): ad libitum
- Acclimation period: min. 3, max. 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21-23 °C
- Humidity (%): ca. 50-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10.0 mL/kg bw

DOSAGE PREPARATION (if unusual): immediately prior administration, emulsion and suspension during application was stirred on a magnetic stirrer

Doses:

681, 1000, 1470, 2150, 3160, 4640 and 10000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: < 15 min, 15 min, 30 min, 1 h, 2 h, 4 h, 5 h and 24 h after application, then daily at least 1 time until day 14 after application, body weight: prior to administration
- Necropsy of survivors performed: yes

Statistics:

Determination of LD50-value: Dose-lethality-time relationship, Probit-Analysis according to FINNEY separated for male and female rats and together for 14-day observation time

Sex:

male

Dose descriptor:

LD50

Effect level:

2 090 mg/kg bw

Based on:

test mat.

95% CL:

>= 1 650 - <= 2 660

Sex:

female

Dose descriptor:

LD50

Effect level:

2 050 mg/kg bw

Based on:

test mat.

95% CL:

>= 1 590 - <= 2 650

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 060 mg/kg bw

Based on:

test mat.

95% CL:

>= 1 720 - <= 2 470

Mortality:

See Table 1 in box "Any other information on results incl. tables".

Clinical signs:

0.681 g/kg: urine dyed blue-green, reduced amount of feces
1.00 to 1.47 g/kg: urine dyed blue-green, reduced amount of feces, apathy, breathing in bursts, breathing snapping and partially wheezing
2.15 to 10.0 g/kg: symptoms started immediately until 30 min after application: apathy, eyelids narrow to closed, lifted fur, breathing snapping, intermittently and gasping, reduced muscle tone, reduced feces, urine blue-green stained, in some rats a strongly distended abdomen was observed. The majority of survived rats was without findings 3 days after application.

Body weight:

No effects observed, normal body weight gain development

Gross pathology:

Spontaneously died rats: systemic blue staining of all organs.
Killed rats at the end of observation period: without findings.

Table 1: Mortality

Doses g/kg	Application volume mL/kg	Concentration mg/mL	1 h		24 h		48 h		7 days		14 days
			male	female	male	female	male	female	male	female	male	female
0.681	10.0	68.1	-	0	-	0	-	0	-	0	-	0
1.00	10.0	100	0	0	0	0	0	1	0	1	0	1
1.47	10.0	147	0	0	0	1	0	1	1	1	1	1
2.15	10.0	215	0	0	0	1	2	2	4	2	4	2
3.16	10.0	316	0	0	0	1	2	3	3	4	3	4
4.64	10.0	464	0	0	5	4	5	5	5	5	5	5
10.0	10.0	909	-	4	-	5	-	5	-	5	-	5

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In conclusion, in an acute oral toxicity study the LD50 value was 2090 mg/kg bw for male rats, 2050 mg/kg bw for female rats and 2060 mg/kg bw for both sexes. By extrapolating the concentration of 20% of the target substance within the mixture to 100% the calculated LD50 value would be 412 mg/kg bw for both sexes and classification as Acute Tox 4, H302 is warranted in accordance with the CLP regulation.
 

Executive summary:

In an acute oral toxicity study, groups of fasted Sprague-Dawley rats (5/sex) were given a single oral dose of the test item (20% purity) suspended in water at doses of 0.681, 1.00, 1.47, 2.15, 3.16, 4.64 and 10.0 g/kg bw and were observed afterwards for 14 days. Symptoms as reduced feces amount, apathy, abnormal breathing was observed at lower doses. In higher doses (2.15 to 10.0 g/kg) symptoms started immediately until 30 min after application. Apathy, eyelids narrow to closed, piloerection, breathing snapping, intermittently and gasping, reduced muscle tone, reduced feces, urine blue-green stained and in some rats a strongly distended abdomen was observed. The majority of survived rats was without findings 3 days after application. No effects in body weight gain was observed. There were no pathological findings except the blue colouring of all organs in spontaneously died rats.

Based on the mortality, the LD50 value was determined with 2090 mg/kg bw for male rats, 2050 mg/kg bw for female rats and 2060 mg/kg bw for both sexes.

By extrapolating the concentration of 20% of the target substance within the mixture to 100% the calculated LD50 value would be 412 mg/kg bw for both sexes and classification as Acute Tox 4, H302 is warranted in accordance with the CLP regulation.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

412 mg/kg bw

Quality of whole database:

Study conducted equivalent/similar to OECD TG 401

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration of exposure:

h

Sex:

male/female

Dose descriptor:

LC0

Effect level:

1.041 mg/L air

Based on:

test mat.

Exp. duration:

1 h

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.041 mg/L air

Based on:

test mat.

Exp. duration:

1 h

Mortality:

No mortality observed

Clinical signs:

other: no signs of toxicity observed

Body weight:

The body weight gain was subnormal in both males and females.
males: 40.7 g (30-57 g)
females: 18.6 g (10-24 g)

Gross pathology:

not specified

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, the LC50 of the test item is greater than 1.0408 mg/L.

Executive summary:

In an acute inhalation toxicity study, groups of young adult Osborne-Mendel rats (5/sex) were exposed by inhalation route to the test item in air for 1 hour to the whole body at a mean concentration of 1.0408 mg/L. Animals then were observed for 14 days. There were no treatment related clinical signs nor mortality, though the body weight gain was sub normal. Based on these results, the LC50 of the test item is greater than 1.0408 mg/L.

The test item is considered to be of low toxicity and does not warrant for classification.

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

1 040.8 mg/m³

Quality of whole database:

Comparable to guideline study with acceptable restrictions

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an acute oral toxicity study (Grundler, 1981) conducted with the target substance (18.3%) in a mixture, the combined oral LD50 was determined to be 3680 mg/kg bw. By extrapolating the concentration of 18.3% of the target substance within the mixture to 100% the calculated LD50 value would be 675 mg/kg bw.

In a second acute oral toxicity study (Hackenberg, 1977) conducted with the target substance (20%) in a mixture, the combined oral LD50 was determined to 2060 mg/kg bw. By extrapolating the concentration of 20% of the target substance within the mixture to 100% the calculated LD50 value would be 412 mg/kg bw for both sexes.

Supporting information is provided with the study by Katz, 1970 in a read-across approach to get information of the toxicity of the chromophore itself. In this study, the acute oral LD50 in rats was determined to be 3700 mg/kg bw in males and 4300 mg/kg bw in females. Details on the read-across rational are provided in section 13.

No data regarding the target substance was available for acute toxicity via the inhalation route. Thus, available data from the chloride salt of the target substance was used in a read-across approach solely to get information on the toxicity of the chromophore itself. Based on the results obtained from an in vivo acute inhalation study, the combined LC₀value of the source substance can be considered to be 1.0408 mg/L. As the substance is only marketed as an aqueous solution containing significant concentrations of free acetic acid as stabiliser, this route of exposure is not relevant and animal testing is not considered to be in accordance with animal welfare regulations.

Justification for classification or non-classification

Based on the available data, the target substance does warrant classification as Acute Tox. 4, H302 in accordance to regulation (EC) No 1272/2008.