3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

Endpoint summary

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Sept 2001 - 18 Oct 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 22,1996

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 30, 1996

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

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 SYSTEM
Source: RCC Ltd; Biotechnology and Animal Breeding Division; CH-4414 Füllinsdod / Switzerland
Number of animals per group: 3 males or 3 females
Total number of animals: 3 males and 3 females
Age when treated: Males: 8 weeks; Females: 10 weeks
ldentification: Unique cage card and corresponding color-coded spots on the tail.
Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Room number: E16
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with target ranges for temperature 22 + 3"C and for relative humidity between 30-70%. 12 hours fluorescent light/12 hours
dark (light period between 6.00 and 18.00), music during the light period.

Accommodation: ln groups of three per sex in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

Diet: Pelleted standard Provimi Kliba 3433 raVmouse maintenance diet, batch no.73/01 (Provimi Kliba AG, CH-4303 Kaiseraugst Switzerland) ad libitum. Results of analyses for contaminants are archived at RCC Ltd, Itingen.

Water: Community tap-water, from ltingen ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300 from Fluka Chemie AG, CH-9471 Buchs (batch number: 42471811 42701)

Details on oral exposure:

VEHICLE
- 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 performed before experimental starting date.
- Lot/batch no. (if required): Supplier: Fluka Chemie AG, CH-9471 Buchs (Batch: 42471811 42701)
- Purity: not specified

MAXIMUM DOSE VOLUME APPLIED: 2000 mg test item/kg bw

DOSAGE PREPARATION (if unusual): The preparations were made shortly before each dosing. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer. 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 oral dose of the test item by gavage at 2000 mg/kg body weight after being fasted for 16 to 20 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing.

Doses:

0: 2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing:
Mortality / Viability Daily during acclimatization and twice daily during days 1-15.
Body weights On test days 1 (pre-administration), I and 15.
- Necropsy of survivors performed: yes
All surviving animals were killed at the end of the observation period by an intraperitoneal injection of NARCOREN (Rhône Mérieux GmbH, D-88471 Laupheim) 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 pedormed. No organs or tissues were retained.
- Other examinations performed: clinical signs: Daily during acclimatization and at least four times on test day 1 after the test item administration. Once daily during days 2-15. All abnormalities were recorded.

Statistics:

No statistical analysis was used.

Preliminary study:

Not performed

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No death occurred during the study.

Clinical signs:

No clinical signs were noted 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 observed at necropsy.

Body weight evolution

Dose [mg/kg]	Animal No.	Sex	Body weight [g]
			Day 1 (treatment)	Day 8	Day 15
2000	1	F	181 .6	200.7	209.2
2000	2	F	178.1	199.3	209.1
2000	3	F	178.7	197.4	207.7
2000	4	M	195.8	243.0	270.6
2000	5	M	208.0	261.7	284.9
2000	6	M	204.7	257.2	284.9

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LDso (rat) of BLUE GS 5664.80 is > 2000 mg/kg bw in test performed according to OECD TG 423 and following GLP.

Executive summary:

The acute oral toxicity of BLUE GS 5664.80 has been determined in the current study performed following OECD TG 423 according to GLP.

One group of three male or three female HanBrl: WIST (SPF) rats was treated by oral gavage with BLUE GS 5664.80 at 2000 mg/kg body weight. The test item was suspended in vehicle (PEG 300) at a concentration of 0.2 g/ml and administered at a volume of 10 ml/kg.

The animals were examined for clinical signs daily during the acclimatization period, four times during test day 1 and once daily during test days 2-15. Mortality/viability was recorded daily during the acclimatization period and together with clinical signs at the same time intervals on test day 1 and twice daily on test days 2-15. Body weights were recorded on day 1 (priorto administration) and on days 1 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study period.

No clinical signs were evident 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 observed at necropsy.

The median lethal dose of BLUE GS 5664.80 after single oral administration to rats of both sexes, observed over a period of 14 days is:

LDso (rat): > 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Klimisch code 1

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001, September 20 to 2001 October 11, 2001

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted February 24, 1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992.

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: HanBrl: WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Source: RCC Ltd; Biotechnology and Animal Breeding Division; CH-4414 Füllinsdod / Switzerland
Number of animals per group: 5 males or 5 females
Total number of animals: 5 males and 5 females
Age when treated: Males: 9 weeks; Females: 11 weeks
ldentification: Unique cage card and corresponding color-coded spots on the tail.
Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Room number: E16
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with target ranges for temperature 22 + 3"C and for relative humidity between 30-70%. 12 hours fluorescent light/12 hours
dark (light period between 6.00 and 18.00), music during the light period.

Accommodation: ln groups of three per sex in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

Diet: Pelleted standard Provimi Kliba 3433 raVmouse maintenance diet, batch no.73/01 (Provimi Kliba AG, CH-4303 Kaiseraugst Switzerland) ad libitum. Results of analyses for contaminants are archived at RCC Ltd, Itingen.

Water: Community tap-water, from ltingen ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen.

Type of coverage:

semiocclusive

Vehicle:

polyethylene glycol

Details on dermal exposure:

TEST SITE
- Area of exposure: back
- % coverage: 10
- Type of wrap if used: The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the skin was flushed with lukewarm tap water and dried with disposable paper towels.
- Time after start of exposure: 24h after the application

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): 0.33 g test item /mL vehicle
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 6mL (vehicle + test item)/kg
- Concentration (if solution): 0.33 g test item /mL vehicle
- Lot/batch no. (if required): Supplier: Fluka Chemie AG, CH-9471 Buchs (Batch: 42471811 42701)
- Purity: not specified

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not specified

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.

- Necropsy of survivors performed: yes
At the end of the observation period all animals were sacrificed by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of at least 2.0 ml/kg body weight (equivalent to at least 320 mg sodium pentobarbitone/kg body weight). The animals were examined macroscopically and all abnormalities recorded. Thereafter, they were discarded.

- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:

Body weights: On test days 1 (pre-administration), 8 and 15.
Clinical signs: Daily during acclimatization and at least four times on test day 1 after the test item administration. Once daily during days 2-15. All abnormalities were recorded.

Statistics:

No statistical analysis was used.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

No deaths occurred during the study.

Clinical signs:

Blue skin and test item residuals were noted in all males on test day two until test day six.
Test item residuals were noted in all females on test day two until test day six and blue skin was observed until test day twelve.

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 observed at necropsy.

Conclusions:

The dermal LDso (rat) of BLUE GS 5664.80 is > 2000 mg/kg bw in test performed according to OECD TG 402 and following GLP.

Executive summary:

The acute dermal toxicity of BLUE GS 5664.80 has been determined in the current study performed following OECD TG 402 according to GLP.

A group of five male and five female HanBrl: WIST (SPF) rats was treated with BLUE GS 5664.80 at 2000 mg/kg by dermal application. The test item was diluted in vehicle (PEG 300) at a concentration of 0.33 g/ml and administered at a volume of 6 ml/kg. The animals were examined for clinical signs four times during test day 1 and once daily during test days 2-15.

Mortality/viability was recorded together with clinical signs at the same time intervals on test day 1. During test days 2-15 it was recorded two times a day. Body weights were recorded on day 1 prior to administration and on days 8 and 15. All animals were necropsied and examined macroscopically.

No deaths occurred during the study.

Blue skin and test item residuals were noted in all males on test day two until test day six.

Test item residuals were noted in all females on test day two until test day six and blue skin was observed until test day twelve.

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

No macroscopic findings were observed at necropsy.

The median lethal dose of BLUE GS 5664.80 after single dermal administration to rats of both sexes, observed over a period of 14 days is:

LDso (rat): > 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Klimisch code 1

Justification for classification or non-classification

For each route of exposure, the LD50/LC50 is > threshold dose leading to classification according to CLP criteria.