disodium 4-hydroxy-3-[2-[4-({4-[2-(1-hydroxy-4-sulfonatonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-05-13 to 2013-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

EC Number:

700-924-0

Molecular formula:

C41H30N4Na2O10S2

IUPAC Name:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental 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

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

cotton seed oil

Details on dermal exposure:

Preparation of the Animals:
The animals were marked for individual identification by tail painting.
Approximately 24 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.

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 cottonseed oil

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:

5 male and 5 female

Control animals:

not required

Details on study design:

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:
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. Attention was directed to observations of tremors, convulsions,
salivation, diarrhoea, lethargy, sleep and coma.

Pathology:
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally
(Narcoren®, Merial) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy. All gross pathological changes
were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation. The preserved tissues of which
no histopathological evaluation was made will be discarded 3 months after the release of the final report unless otherwise agreed upon with
the sponsor.

Evaluation of Results:
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.

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

Dose descriptor:

approximate LD50

Effect level:

> 2 000

Mortality:

No mortality was observed.

Clinical signs:

No treatment-related effects were observed.

Body weight:

A slight weight loss was recorded for 3 out of 5 female animals during the first week, but all of these female animals showed weight gain
during the second week except for animal no. 30, which showed also a weight loss during the second week.
one animal showed a slight weight loss during the second week, but nevertheless an overall positive body weight development.
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.

Gross pathology:

No treatment-related effects were observed.

Other findings:

No erythema or oedema was observed. Crust was observed in 1 of 5 male animals. Scratches were observed in 2 of 5 male and 2 of 5 female animals.
The signs of irritation persisted until the end of the observation period in 2 out of 5 female animals.

Any other information on results incl. tables

Table 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

Table 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	during the whole observation period	no signs of toxicity

bw = body weight

Table 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	cs, *	0/0	*	0/0	*	0/0	*	0/0	*
day 7	0/0	cs, *	0/0	*	0/0	*	0/0	*	0/0	*
day 8	0/0	nsf	0/0	s,*	0/0	nsf	0/0	s	0/0	nsf
day 9	0/0	nsf	0/0	*	0/0	nsf	0/0	s	0/0	nsf
day 10	0/0	nsf	0/0	*	0/0	nsf	0/0	s	0/0	nsf
day 11	0/0	nsf	0/0	*	0/0	nsf	0/0	s	0/0	nsf
day 12	0/0	nsf	0/0	*	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
Comments: E = erythema; O = oedema; 0, 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) * = residue of test item (red); cs = crust; s = scratches; nsf = no specific findings

Table 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	*	0/0	*
day 5	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 6	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 7	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 8	0/0	nsf	0/0	s	0/0	*	0/0	*	0/0	s, *
day 9	0/0	nsf	0/0	s	0/0	*	0/0	*	0/0	s, *
day 10	0/0	nsf	0/0	s	0/0	*	0/0	*	0/0	s, *
day 11	0/0	nsf	0/0	s	0/0	*	0/0	*	0/0	s, *
day 12	0/0	nsf	0/0	s	0/0	*	0/0	*	0/0	s, *
day 13	0/0	nsf	0/0	s	0/0	nsf	0/0	nsf	0/0	s
day 14	0/0	nsf	0/0	s	0/0	nsf	0/0	nsf	0/0	s
day 15	0/0	nsf	0/0	s	0/0	nsf	0/0	nsf	0/0	s
Comments: E = erythema; O = oedema; 0, 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) * = residue of test item (red); s = scratches; nsf = no specific findings

Table 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	233	254	280	20
22/male	241	247	280	16
23/male	240	267	300	25
24/male	238	253	278	17
25/male	240	259	292	22
26/female	202	203	202	0
27/female	213	204	212	0
28/female	206	209	214	4
29/female	209	206	210	0
30/female	215	213	211	-2

Table 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

Table LD50:

Dose (Unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50
2000 mg/kg bw	5 males	0	> 2000 mg/kg bw
2000mg/kg bw	5 females	0	> 2000 mg/kg bw

bw = body weight

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

Under the conditions of the present study, single dermal application of the test item Acid Red RN 2949 to rats a dose of
2000 mg/kg body weight was associated with neither mortality nor signs of toxicity, but signs of irritation.
The dermal LD50 was determined to be > 2000 mg Acid Red RN 2949 / kg body weight.

Executive summary:

Summary Results

LD₅₀: 2000 mg /kg bw

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

Vehicle (moistening): cottonseed oil

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

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:

No treatment-related effects were observed.

Effect on organs (related to dose level): No treatment-related effects were observed.

Signs of irritation: No erythema or oedema was observed. Crust was observed in 1 of 5 male animals. Scratches were observed in 2 of 5 male and 2 of 5 female animals.

The signs of irritation persisted until the end of the observation period in 2 out of 5 female animals.

Conclusion

Under the conditions of the present study, single dermal application of the test item Acid Red RN 2949 to rats a dose of 2000 mg/kg body weight was associated with neither mortality nor signs of toxicity, but signs of irritation.

The dermal LD50 was determined to be > 2000 mg Acid Red RN 2949 / kg body weight.