Reaction mass of isomers disodium 8-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-5-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, disodium 5-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-8-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-07 to 2013-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

1

Reference substance name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

EC Number:

700-946-0

Molecular formula:

C40H35N7Na2O6S2

IUPAC Name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1025)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups / individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 011012)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The animals were marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted).
Following the period of fasting the animals were weighed and the test item was administered.
Food was provided again approximately 4 hours post dosing.
The test item was administered at a single dose by gavage using a feeding tube.
The test item was administered at a dose volume of 10 mL/kg body weight.

Doses:

The starting dose was selected to be 2000 mg/kg body weight. No compound-related mortality was recorded for any animal of step 1 or 2.
Based on these results and according to the acute toxic class method regime no further testing was required.

No. of animals per sex per dose:

3 animals/step

Control animals:

no

Details on study design:

Observation Period
All animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality.

Weight Assessment
The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.

Clinical Examination
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology
At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally (Narcoren®, Merial; lot no.: 224052; expiry date: 05/2015) at a dosage of approximately 8 mL/kg bw.
All animals were subjected to gross necropsy. All gross pathological changes were recorded.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

All animals survived until the end of the study.

Clinical signs:

other: The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection and half eyelid-closure. All symptoms recovered within up to 4 hours post dose.

Gross pathology:

At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

Any other information on results incl. tables

Table: Clinical Signs - Individual Data

Animal No. / Sex	Time of Observation Post-Dose	Observations
Step 1 (2000 mg/kg Body Weight)
1 / female,2 / female, 3 / female	1 h, 2 h, 3 h	slightly reduced spontaneous activity, slight piloerection, half eyelid closure
	4 h until the end of the observation period	no signs of toxicity
Step 2 (2000 mg/kg Body Weight)
4 / female, 5 / female, 6 / female	1 h	slightly reduced spontaneous activity
	2 h, 3 h	slightly reduced spontaneous activity, slight piloerection
	4 h until the end of the observation period	no signs of toxicity

d = day (day 1 = day of administration);     h = hour(s);       min = minute(s)

Table: Body Weight Development - Absolute Body Weights in g and Body Weight Gain in %

Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
Step 1 (2000 mg/kg Body Weight)
1 / female	168	187	194	15
2 / female	173	202	211	22
3 / female	182	104	203	12
Step 2 (2000 mg/kg Body Weight)
4 / female	156	185	183	17
5 / female	148	178	189	28
6 / female	160	193	200	25

Table: Findings of Necropsy - Individual Data

Animal No./ Sex	Organ	Macroscopic Findings
Step 1 (2000 mg/kg Body Weight)
1 / female	-	nsf
2 / female	-	nsf
3 / female	-	nsf
Step 2 (2000 mg/kg Body Weight)
4 / female	-	nsf
5 / female	-	nsf
6 /female	-	nsf

nsf = no specific findings

Table: LD50 Cut-Off

Dose (unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50 Cut-Off
2000 mg/kg bw	6	0	> 2000 mg/kg bw

bw = body weight

Applicant's summary and conclusion

Interpretation of results:

other: not classified as harmful or toxic according to the CLP Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the present study, a single oral application of the test item Acid Black RN 3333 to rats at a dose of 2000 mg/kg bw was associated with no signs of toxicity or mortality.
The median lethal dose of test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 female (rat): > 2000 mg/kg bw.

Executive summary:

 

Summary Results

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight.

The test item was suspended in the vehicle aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days.

The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs.

All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15.All animals were necropsied and examined macroscopically.

  Results per Step

Step	Sex/No.	Dose (mg/kg)	Number of Animals	Number of Intercurrent Deaths
1	female/1-3	2000	3	0
2	female/4-6	2000	3	0

All animals survived until the end of the study showing signs of toxicity.

The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection and half eyelid-closure. All symptoms recovered within up to 4 hours post dose.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

LD₅₀cut-off:                                                     > 2000 mg/kg bw

Species/strain:                                                   WISTAR Crl: WI(Han) rats

Number of animals:                                           3 per step / 2 steps performed

Vehicle:                                                            aqua ad injectionem

Method:                                                             OECD 423, EC 440/2008, Method B.1 tris, OPPTS 870.1100

 

 Conclusion

Under the conditions of the present study, a single oral application of the test item Acid Black RN 3333 to rats at a dose of 2000 mg/kg body weight was associated with no signs of toxicity or mortality.

The median lethal dose of Acid Black RN 3333 after a single oral administration to female rats, observed over a period of 14 days is: LD₅₀female (rat): > 2000 mg/kg bw