Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-24 to 2013-09-09

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:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

EC Number:

700-936-6

Molecular formula:

C17H11F2N7Na2O7S2

IUPAC Name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

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. 1531)
- 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:

water

Remarks:

AlleMan Pharma, lot no. 26210S1-2, expiry date: 09/2015

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 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:

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; lot no. 224052; expiry date: 05/2015) 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

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed

Clinical signs:

No treatment-related effects were observed.

Body weight:

A slight weight loss was recorded for 1 out of 5female 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.

Gross pathology:

No treatment-related effects were observed.

Other findings:

Neither erythema nor oedema were observed in any animal throughout the observation period.
Eschar and scratches were observed in 1 of 5 male animals.
These signs of irritation were reversible within the observation period.
Test item residues observed in all tested animals were not assumed to be a sign of irritation.

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	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 3	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 4	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 5	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 6	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 7	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 8	0/0	tir	0/0	tir, s	0/0	tir	0/0	c	0/0	tir
day 9	0/0	tir	0/0	tir, es	0/0	tir	0/0	c	0/0	tir
day 10	0/0	tir	0/0	tir, es	0/0	tir	0/0	c	0/0	tir
day 11	0/0	tir	0/0	tir	0/0	tir	0/0	c	0/0	tir
day 12	0/0	tir	0/0	tir	0/0	tir	0/0	c	0/0	tir
day 13	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
day 14	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
day 15	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
Comments: E = erythema; O = oedema; 0, 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) tir = test item residues; s = scratches above and in the center of the application site; es = eschar above and in the center of the application site; reversibility: c = completely reversed

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	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 3	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 4	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 5	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 6	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 7	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 8	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 9	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 10	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 11	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 12	0/0	tir	0/0	tir	0/0	tir	0/0	tir	0/0	tir
day 13	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
day 14	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
day 15	0/0	c	0/0	c	0/0	c	0/0	c	0/0	c
Comments: E = erythema; O = oedema; 0, 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) tir = test item residues; reversibility: c = completely reversed

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	248	260	292	18
22/male	249	271	307	13
23/male	240	248	284	18
24/male	238	253	290	22
25/male	242	256	293	21
26/female	215	215	220	2
27/female	210	211	228	9
28/female	216	208	216	0
29/female	223	226	236	6
30/female	208	209	219	5

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

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:

other: LD 50 > 2000 mg/kg bw

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

Under the conditions of the present study, single dermal application of the test item Reactive Golden Yellow HF-RN 1331 to rats at a dose
of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity but slight signs of irritation in one out of ten
animals which were reversible within the observation period.
The dermal LD50 was determined to be > 2000 mg Reactive Golden Yellow HF-RN 1331 / kg body weight.

Executive summary:

Summary Results

LD₅₀: > 2000 mg /kg bw

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

Vehicle (moistening): aqua ad injectionem

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

Table:  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: Neither erythema nor oedema were observed in any animal throughout the observation period. Eschar and scratches were observed in 1 of 5 male animals. These signs of irritation were reversible within the observation period. Test item residues observed in all tested animals were not assumed to be a sign of irritation.

Conclusion

Under the conditions of the present study, single dermal application of the test item Reactive Golden Yellow HF-RN 1331 to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity but slight signs of irritation in one out of ten animals which were reversible within the observation period.

The dermal LD50 was determined to be > 2000 mg Reactive Golden Yellow HF-RN 1331 / kg body weight.