disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 26, 2002 - May 14, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 22, 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 30, 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-050-5

EC Name:

-

IUPAC Name:

dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid

Details on test material:

Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

Test animals

Species:

rat

Strain:

other: HanBrl: Wist (SPF)

Remarks:

recognized by the international guidelines as a recommended test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Biotechnology and Animal Breeding Division CH-4414 Füllinsdorf / Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age when treated: 8 weeks (males), 10 weeks (females)
- Weight prior to administration: 163 g (female #1), 154.2 g (female #2), 159.2 g (female #3), 242.4 g (male #4), 221.0g (male #5), 235.9 g (male #6)
- Fasting period before study: 18-20 hours (access to water was permitted)
- Housing: in groups of three per sex in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ('Lignocel' Schill AG, CH-4132 Mutten/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3433 rat maintenance diet, batch no. 119/01 (Provimi Kliba AG, CH-4303 KaiseraugsV Switzerland) ad libitum. Results of analyses for contaminants are archived at RCC Ltd, Itingen.
- Water (e.g. ad libitum): community tap-water, from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen.
- Acclimation period: under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 30-70% (values above 70% during cleaning process possible)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (music during the light period)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Purified water

Remarks:

deionised water which was processed and treated by the PURELAB Option-R unit. The vehicle was chosen after a non-GLP solubility trial (trial formulation excluded from the GLP statement of compliance)

Details on oral exposure:

VEHICLE: purified water
- Concentration in vehicle: 0.2g test item/ml vehicle
- Amount of vehicle (if gavage): 10 ml vehicle/kg bw
- Justification for choice of vehicle: The vehicle was chosen after a non-GLP solubility trial which was performed before the study initiation date. This trial formulation is excluded from the GLP statement of compliance.
- Lot/batch no. (if required): not detailed
- Purity: purified water

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg body weight

DOSAGE PREPARATION (if unusual): The dose formulations were made shortly before each dosing occasion. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The formulations were prepared using first a glass stick and then a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: limit concentration of 2000 mg/kg bw

TREATMENT
The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg body weight after being fasted for 18 to 20 hours (access to water was permitted). Food was provided again 3 hours after dosing.

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
Mortality / Viability: daily during acclimatization and twice daily during days 1-15
Body weights : on test days 1 (prior to administration), 8 and 15
Clinical signs: daily during acclimatizalion and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities were recorded.

- Necropsy of survivors performed: yes
All animals were killed at the end of the obseruation period by an intraperitoneal injection of NARCOREN at a dose of at least 2.0 ml/kg body weight (equivalent to at least 320 mg sodium pentobarbitone/kg body weight) and discarded after macroscopic examinations were peformed. No organs or tissues were retained.

- Other examinations performed: -

Statistics:

No statistical analysis was perfomed.

Results and discussion

Preliminary study:

Not performed

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred during the study.

Clinical signs:

No clinical signs were obserued during the course of the study.

Body weight:

The body weight of the animals was within the range commonly recorded for this strain and age.

Gross pathology:

No macroscopic findings were recorded at necropsy.

Any other information on results incl. tables

Evolution of body weight (in grams):

Sex/dose	Animal No.	Day 1 (= day of treatment)	Day 8	Day 15
Female / 2000 mg/kg	1	163.0	183.0	198.5
	2	154.2	176.3	188.1
	3	159.2	175.8	177.4
Male / 2000 mg/kg	4	242.4	277.1	312.0
	5	221.0	253.0	277.3
	6	235.9	278.3	308.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50(rat) of Red RWa 4565 is > 2000 mg/kg bw in test performed according to OECD TG 423 and following GLP.

Executive summary:

The acute oral toxicity of Red RWa 4565 has been determined in the current study performed following OECD TG 423 according to GLP.

One group of three male and three female HanBrl: WIST (SPF) rats was treated with Red RWa 4565 by oral gavage administration at a dosage of 2000 mg/kg body weight.

The test item was diluted in vehicle (purified water) at a concentration of 0.2 g/ml and administered at a volume dosage of 10 ml/kg.

 

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs at approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded twice daily during test days 1-15. Body weights were recorded on day 1 (prior to administration) and on days I and 15. All animals were necropsied and examined macroscopically.

 

The following animals were treated at 2000 mg/kg bw and percentage of mortality was observed: males 0% and females 0%

 

All animals survived until the end of the study period.

No clinical signs were observed during the course of the study.

The body weight of the animals was within the range commonly recorded for this strain and age.

No macroscopic findings were recorded at necropsy.

The median lethal dose of Red RWa 4565 after single oral administration to rats of both sexes, observed over a period of 14 days is:

LD₅₀(RAT): GREATER THAN 2000 MG/KG BODY WEIGHT