1,3,6-Naphthalenetrisulfonic acid, 7-[2-[2-[(aminocarbonyl)amino]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]phenyl]diazenyl]-, sodium salt

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2007 to 23 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

474-870-9

EC Name:

-

Cas Number:

80156-97-4

Molecular formula:

Hill formula: C28H20ClN9Na4O16S5 CAS formula: C28H24ClN9O16S5.4Na

IUPAC Name:

tetrasodium 7-[(1E)-2-[2-(carbamoylamino)-4-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl]naphthalene-1,3,6-trisulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Yellow 176 Ester
Reactive Yellow 176 Sulfato

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species and strain: CRL:(WI) BR rats
Source: CHARLES RIVER (EUROPE) LABORATORIES INC. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
Justification of strain: The Wistar rat as a rodent is one of the standard animal in toxicity studies.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Number of animals: 6 animals, 3 animals/group
Sex: Female, nulliparous and non-pregnant.
Age of animals: Young healthy adult rats, less than 10 weeks old
Date of receipt: 03 May 2007
Body weight at treatment: Between range of 184 g and 188 g
Acclimatization time: 5 and 6 days (Group 1 and Group 2)
Animal health: Only healthy animals were used for the test. The veterinarian certified healthy status.
Number of animal room: 245-8
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding: Laboratory bedding
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 8-12 air exchanges/hour by central air-condition system.
Food and Water Supply: The animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494
Soest Germany, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal Identification The individual identification was performed by numbers on the tail written with a marker pen. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

Vehicle
Name: Distilled water
Batch No: 8991006
Expiry Date: October 2009
Supplier: Humantrade Ltd.
Storage: At room temperature

Formulation
For treatment the test item was applied in a concentration of 200 mg/ml in distilled water. Formulations were prepared just before the administration and stirred with a magnetic stirrer up to end of the treatment.

Doses:

Doses
Justification of the dose:
Starting dose was selected on the basis of the information provided by the Sponsor.
The LD50 value was expected to be above 2000 mg/kg bw. A limit test was performed at 2000 mg/kg bw dose.
Three female animals were treated with a dose level of 2000 mg/kg bw of Gelb Sulfato in the first step.

No. of animals per sex per dose:

6 animals all female, 3 animals/group

Control animals:

yes

Details on study design:

A single oral administration - followed by a fourteen-day observation period – was performed by gavage. On the day before each treatment the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before the treatment. The test item was administered by oral gavage in the morning hours. The food was given back 3 hours after the treatment. A constant treatment volume of 10 ml/kg body weight was applied.

Clinical Observations
Careful clinical observation was made 15 and 30 minutes, 1 h, 2 h, 3 h, 4 h, 6 h after the treatment and once each day for 14 days thereafter.
Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern as well. Particular attention was directed to the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Body Weight Measurement
The body weight was recorded on day 0 (shortly before the treatment), then on days 7 and 14 with precision of 1g.

NECROPSY
Gross necropsy was performed in each experimental animal terminally. Animals were sacrificed by exsanguination under pentobarbital anaesthesia. After the examination of the external appearance the cranial, thoracic and the abdominal cavities were opened, the organs and the tissues were observed.
Abnormalities were recorded on post mortem data sheets.

Statistics:

No data

Results and discussion

Preliminary study:

Three female animals were treated with a dose level of 2000 mg/kg bw of Gelb Sulfato in the first step. All animals survived; so three further animals were dosed at 2000 mg/kg bw dose level next day, as the second step. No mortality occurred in either steps, therefore the test was finished meeting the stopping criteria of guidelines (OECD 423, Directive 2004/73/EC B.1.tris, OPPTS 870.1100).

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Gelb Sulfato caused no mortality in female CRL:(WI) BR rats after a single oral (by gavage) administration of 2000 mg/kg bw.

Clinical signs:

other: Group 1 – 2000 mg/kg bw Slight activity decrease, hunched back and piloerection were noted for one animal (1/3) two hours after the treatment. There were no test item effect on the physical condition and behaviour of the remaining animals (2/3). Yellow co

Gross pathology:

No macroscopic alterations related to the toxic effect of Gelb Sulfato were found at necropsy. Hydrometra (1/3, Group 1) is a common necropsy findings in experimental rats related to the sexual cycle of animals. The pinprick-sized haemorrhages (1/3 in Group 2) observed in the lungs were due to the exsanguination.

Other findings:

None

Any other information on results incl. tables

CLINICAL OBSERVATIONS

 

SUMMARY OF OBSERVATION

Observations	Frequency of symptoms
	Group 1 2000 mg/kg bw	Group 2 2000 mg/kg bw
Normal	2/3	3/3
Decreased activity	1/3	0/3
Hunched back	1/3	0/3
Piloerection	1/3	0/3

Comment: Frequency: number of animals with observation / number of animals observed

INDIVIDUAL CLINICAL OBSERVATIONS

Group 1	Animal Number	Observations	Day 0							Day 1-14	F
			15’	30’	1h	2h	3h	4h	6h
2000 mg/kg bw	5638	Normal	+	+	+	+	+	+	+	+	21/21
	5640	Normal	+	+	+	+	+	+	+	+	21/21
	5646	Normal	+	+	+	-	+	+	+	+	20/21
		Deceased activity	-	-	-	+	-	-	-	-	1/21
		Hunched back	-	-	-	+	-	-	-	-	1/21
		Piloerection	-	-	-	+	-	-	-	-	1/21
Group 2	Animal Number	Observations	Day 0							Day 1-14	F
			15’	30’	1h	2h	3h	4h	6h
2000 mg/kg bw	5644	Normal	+	+	+	+	+	+	+	+	21/21
	5645	Normal	+	+	+	+	+	+	+	+	21/21
	5647	Normal	+	+	+	+	+	+	+	+	21/21

Comments: F = Frequency of observation = number of occurrence of observation / total number of observations per animal

              + = Observation present; - = Observation no present;

               ‘ = Minutes

              h = hour

 

NECROPSY FINDINGS

 

TEST ITEM: GELB SULFATO			TEST SYSTEM: CRL: (WI) BR RAT			STUDY CODE: 07/425-001P
MODE OF ADMINISTRATION: ORAL			SEX: FEMALE			DURATION OF STUDY: 14 DAYS
DOSE: 2000 MG/KG BW			NUMBER OF ANIMALS: 3/3			DATE OF NECROPSY: 22 MAY 2007
NECROPSY FINDINGS PER ORGANS	ANIMAL NUMBERS:	5638	5640	5646	/	/	NECROPSY FINIDINGS
							Σ	%
Uterus	Hydrometra	+	-	-	/	/	1	33
No organs with macroscopic findings		-	+	+	/	/	2	66
TEST ITEM: GELB SULFATO			TEST SYSTEM: CRL: (WI) BR RAT			STUDY CODE: 07/425-001P
MODE OF ADMINISTRATION: ORAL			SEX: FEMALE			DURATION OF STUDY: 14 DAYS
DOSE: 2000 MG/KG BW			NUMBER OF ANIMALS: 3/3			DATE OF NECROPSY: 23 MAY 2007
NECROPSY FINDINGS PER ORGANS	ANIMAL NUMBERS:	5644	5645	5647	/	/	NECROPSY FINDINGS
							Σ	%
LUNGS	Pinprick-sized haemorrhages	+	-	-	/	/	1	33
No organs with macroscopic findings		-	+	+	/	/	2	66

COMMENT: NO ALTERATION = -

                     ALTERATION PRESENT = +

                    NO DATA = /

                    GRADE OF ALTERATION 1 = SLIGHT /SMALL / FEW

                                                   2 = MODERATE / M.SIZE / M. NUMBER

                                                   3 = MARKED / MANY / LARGE

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral LD50 value of the test item Gelb Sulfato proved to be greater than 2000 mg/kg body weight in female CRL:(WI) BR rats.

Executive summary:

An acute oral toxicity study was performed to assess the acute toxicity of test item Gelb Sulfato in rats. The results of the study allowed the test item to be ranked according to the classification systems, currently in use.

 

The acute toxic class method according to OECD 423 and Directive 2004/73/EC B.1. tris was performed with Gelb Sulfato. The study was performed in accordance with the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Two groups of three female CRL:(WI) BR Wistar rats were treated by single oral gavage with Gelb Sulfato at dose levels of 2000 mg/kg bw in two independent experiments (Treatment group 1 and Treatment group 2). A concentration of 200 mg/ml prepared with distilled water corresponding to a treatment volume of 10 ml/kg bw was used at both steps.

 

Clinical observations were performed for all animals 15 and 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Food was made available again 3 hours after the treatment. Body weight was measured weekly. Gross necropsy was performed on all animals at termination of examination (day 14).

 

Results

Mortality:No mortality occurred after a single oral administration of Gelb Sulfato at 2000 mg/kg bw dose level in female CRL:(WI) BR rats.

 

Treatment group:	1	2
Dose level (mg/kg bw):	2000	2000
Number of animals treated	3	3
Mortality:	0/3	0/3

 

Clinical symptoms:Slight activity decrease, hunched back and piloerection were noted for one animal (1/6) two hours after the treatment. There were no test item effect on the physical condition and behaviour of the remaining animals. Yellow coloured urine and faces were noted on the day of the treatment and on the next day.

 

Body weight and body weight gain:The mean body weight and the body weight gain of animals were in the normal range during the two weeks observation period, similar to the expected values in untreated animals of the same age and strain.

 

Necropsy

No macroscopic alterations related to the toxic effect of Gelb Sulfato were found

 

Conclusion:

The acute oral LD50value of the test item Gelb Sulfato was greater than 2000 mg/kg body weight in female CRL:(WI) BR rats.

Gelb Sulfato does not meet the requirements of EU labelling regulations.