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: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-08 to 2013-07-04

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 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

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 material form:

solid: particulate/powder

Remarks:

migrated information: 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. 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 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

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 surviving 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 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.

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:

All animals showed no signs of toxicity.

Body weight:

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

Gross pathology:

At necropsy, no 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	during the whole observation period	no signs of toxicity
2 / female	during the whole observation period	no signs of toxicity
3 / female	during the whole observation period	no signs of toxicity
Step 2 (2000 mg/kg Body Weight)
4 / female	during the whole observation period	no signs of toxicity
5 / female	during the whole observation period	no signs of toxicity
6 / female	during the whole observation period	no signs of toxicity

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	143	159	167	17
2 / female	146	163	173	18
3 / female	157	171	184	17
Step 2 (2000 mg/kg Body Weight)
4 / female	148	177	193	30
5 / female	141	165	186	32
6 / female	154	180	194	26

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	LD50Cut-Off
2000 mg/kg bw	6	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, a single oral 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 signs of toxicity or mortality.
The median lethal dose of Reactive Golden Yellow HF-RN 1331 after a single oral administration to female rats, observed
over a period of 14 days is: LD50 (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.

Table:  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 without showing any signs of toxicity.

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 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[3]
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 Reactive Golden Yellow HF-RN 1331 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 Reactive Golden Yellow HF-RN 1331 after a single oral administration to female rats,

observed over a period of 14 days is: LD₅₀(rat)>2000 mg/kg bw