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: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2013-June 2013

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

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:

male/female

Details on test animals or test system and environmental conditions:

Species/strain: healthy rats, WISTAR rats Crl: WI(Han) (full barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female
The female animals were non-pregnant and nulliparous.
Number of animals: 5 male and 5 female
Age at the beginning of the study: males: 8 - 9 weeks old, females: 8 - 9 weeks old
Body weight on the day of administration: males: 226 – 242 g; females: 208 – 213 g.

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
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. 0902)
- 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 individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 240113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Preparation of the Animals
The animals were marked for individual identification by tail painting.
Approximately 25 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper.
Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24 hour period. The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

Duration of exposure:

The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed using aqua ad injectionem

Doses:

The test item was applied at a single dose of 2000 mg/kg body weight to each animal.

No. of animals per sex per dose:

Number of animals: 5 male and 5 female

Control animals:

no

Details on study design:

Preparation of the Animals
The animals were marked for individual identification by tail painting.
Approximately 25 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper.
Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.

Application
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period. The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

Dose Level
The test item was applied at a single dose of 2000 mg/kg body weight to each animal.

Exposure Period
The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed using aqua ad injectionem.

Observation Period
All animals were observed for 14 days after dosing.

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

Clinical Examination
A careful clinical examination was made several times on the day of dosing (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. Attention was directed to observations of tremors, 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. 228112; expiry date: 30/11/2015) at the dosage of approximately 8 mL/kg bw.
All animals were subjected to gross necropsy. All gross pathological changes were recorded.

Results and discussion

Preliminary study:

The test item showed signs of acute dermal toxicity and signs of dermal irritation after a single dose application in one female only.
A slight weight loss was recorded for 4 out of 5 female animals during the first week, but all of the female animals showed weight gain during the second week. The effects on weight development might be secondary to the dressing, and toxicological relevance of this finding cannot clearly be concluded.
The male animals showed weight gain during the first and the second week of the observation.

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No

Clinical signs:

other: Abnormal breathing on day 8 and slight piloerection on some occasions were observed in 1 of 5 female animals.

Gross pathology:

With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Any other information on results incl. tables

Clinical Signs of Systemic Toxicity – Individual Data - Males

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 5)
21/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
22/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
23/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
24/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
25/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

bw = body weight

Clinical Signs of Systemic Toxicity – Individual Data – Females

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 6)
26/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
27/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
28/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
29/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
30/ female / 2000 mg/kg bw	0 min, 30 min, 1 h, 2 h, 3 h, 4 h	no signs of toxicity
	day 2	slight piloerection
	day 3 to day 7	no signs of toxicity
	day 8	slight piloerection; slightly abnormal breething
	day 9 to day 13	slight piloerection
	day 14 until the end of the observation period	no signs of toxicity

min = minute(s), h = hour(s), bw = body weight

Skin Irritation – Individual Data – Males

Day after Start of Application	Animal No. 21		Animal No. 22		Animal No. 23		Animal No. 24		Animal No. 25
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 3	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 4	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 5	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 6	0/0	nsf	0/0	nsf	0/0	*	0/0	*	0/0	*
day 7	0/0	nsf	0/0	nsf	0/0	*	0/0	*	0/0	*
day 8	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 9	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 10	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 11	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 12	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 13	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 14	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 15	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
Comments: E = erythema; O = oedema; * = residue of test item (black) nsf =no specific findings

Skin Irritation – Individual Data – Females

Day after Start of Application	Animal No. 26		Animal No. 27		Animal No. 28		Animal No. 29		Animal No. 30
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 3	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 4	0/0	*	0/0	*	0/0	*	0/0	*, s	0/0	*
day 5	0/0	*	0/0	*	0/0	*	0/0	*, s	0/0	*
day 6	0/0	*	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 7	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 8	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 9	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 10	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 11	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 12	0/0	nsf	0/0	nsf	0/0	nsf	0/0	s	0/0	nsf
day 13	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 14	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 15	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
E = erythema; O = oedema; * = residue of test item (black); s = scratches nsf =no specific findings

Absolute Body Weights in g and Body Weight Gain in %

Dose: 2000 mg/kg body weight
Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
21 / male	238	251	280	17.6
22 / male	235	249	280	19.1
23 / male	242	252	284	17.4
24 / male	226	239	269	19.0
25 / male	240	254	285	18.8
26 / female	208	210	221	6.3
27 / female	209	203	208	-0.5
28 / female	209	202	204	-2.4
29 / female	210	207	215	2.4
30 / female	213	203	213	0

Macroscopic Findings - Individual Data – Males and Females

Dose: 2000 mg/kg bw
Animal No. / Sex	Organ	Macroscopic Findings
21 / male	-	nsf
22 / male	-	nsf
23 / male	-	nsf
24 / male	-	nsf
25 / male	-	nsf
26 / female	-	nsf
27 / female	-	nsf
28 / female	-	nsf
29 / female	-	nsf
30 / female	-	nsf

nsf = no specific findings

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, single dermal application of the test item Acid Black RN 3333 to rats at a dose of 2000 mg/kg body weight was associated with no mortality but signs of toxicity in females which had a slight weight loss during the first week. Abnormal breathing on day 8 and slight piloerection on some occasions were seen in one female. There were no signs of dermal irritation with the exception of one female.
The dermal LD50 was determined to be > 2000 mg Acid Black RN 3333 / kg body weight.

Executive summary:

Acid black RN 3333 at single dose of 2000 mg/kg was applied over an area which was approximately 10% of the total body surface on the dorsal area (fur was removed) of five males and five females WISTAR Crl: WI(Han) rats and held in contact with the skin by a dressing throughout 24-hour period. The vehicle used for moistening was aqua ad injectionem. At the end of the treatment period the test item was removed using aqua ad injectionem and all the animals were observed for 14 days after application. No mortality occurred during the course of the study. For male animals no body weight loss was recorded while for the females some slight body weight loss was observed in two of them and scratches were observed in one female starting on day 4 until day 12 post application. Another female was noted to have slight piloerection on some occasions and abnormal breathing on day 8 post application. No other clinical signs were noted. At necropdy no macroscopic findings were observed.

 

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

Vehicle (moistening):                               aqua ad injectonem (sterile water)

Number of animals:                                  5 male and 5 female

Duration of exposure:                             24 hours

Method:                                                  OECD 402
EC 440/2008, Method B.3
OPPTS 870.1200

Results per Step

Sex	Dose (mg/kg bw)	Number of Animals	Number of Intercurrent Deaths
male	2000	5	0
female	2000	5	0

Signs of toxicity related to dose level used, time of onset and duration:

Abnormal breathing on day 8 and slight piloerection on some occasions were observed in 1 of 5 female animals.

Effect on organs (related to dose level):

No treatment-related effects were observed.

Signs of irritation:

No erythema or oedema was observed. Scratches were observed in 1 of 5 female animals.

All signs of irritation were reversible within the observation period.