Tetra(sodium/potassium)-7-[(E)-{2-acetamido-4-[(E)-(4-{[4-chlor-6-({2-[(4-fluor-6-{[4-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonato-1-naphthyl)diazenyl]-5-methoxyphenyl}diazenyl]-1,3,6-naphthalintrisulfonate

Endpoint summary

Description of key information

LD50 (female, rat, oral): greater than 2000 mg/kg body weight.
LD50 (female/male, rat, dermal): greater than 2000 mg/kg body weight

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:

Experiment start date - 14 June 2006; Experiment completion date - 07 July 2006; Study completion date - 24 August 2006.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Test System: Rat, HanRcc:WIST (SPF)
Number of animals per group: 3 females
Total number of animals: 6 females
Age at treatment: 12 weeks (females)
Identification: By unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: Under laboratory conditions after health examination. Only healthy animals were used for the study.

Conditions
Standard Laboratory Conditions.
Air-conditioned with ranges for room temperature of 22 ± 3 °C and relative humidity 30-70 % and approximately 10-15 air changes per hour. 12 hours artificial fluorescent light/12 hours dark, music during the light period.
Accommodation: during acclimatization in groups of five per sex in Makrolon type-4 cages with standard softwood bedding.
Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet (batch 001/06) ad libitum.
Water: Community tap water, ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg body weight after being fasted for approximately 18 to 19 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing.
The application volume was 10 mL/kg body weight.

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

6 females

Control animals:

no

Details on study design:

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with test item 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 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically. All animals survived until the end of the study period.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Mild or moderate clinical signs of toxicity (ruffled fur, hunched posture, sedation) were noted in the animals on the day of dosing and a slightly ruffled fur persisted in two animals still on day 2. From day 3 on for the remaining observation period all animals were absent of clinical findings.

Clinical signs:

other: A ruffled fur was observed in all animals on the day of dosing starting 1, 2 or 3 hours after dosing and persisting up to the 5-hour reading. This symptom was graded moderate in two animals 2-5 or 3-5 hours after dosing and otherwise considered slight. A

Gross pathology:

No macroscopic findings were recorded at necropsy.

Interpretation of results:

not classified

Conclusions:

LD50 (female rat, oral): greater than 2000 mg/kg body weight.

Executive summary:

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with test item 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 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically. All animals survived until the end of the study period. Mild or moderate clinical signs of toxicity (ruffled fur, hunched posture, sedation) were noted in the animals on the day of dosing and a slightly ruffled fur persisted in two animals still on day 2. From day 3 on for the remaining observation period all animals were absent of clinical findings. The body weight of the animals was within the range commonly recorded for this strain and age. No macroscopic findings were recorded at necropsy. Therefore, the LD50 of test item can be estimated to be greater than 2000 mg/kg bw, which indicates that test article shall not be classified according to CLP (Regulation EC No. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

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:

Experiment start date - 13 June 2006; Experiment completion date - 04 July 2006; Study competion date - 29 August 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Identity: FAT 40827/A
Batch: T2 5572 BOP 01/06
Purity: determined in this study
Appearance: black sticky powder
Expiration date: 28.02.2011
Storage: at room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat, HanRcc:WIST (SPF)
Number of animals per group: 5 males and 5 females
Total number of animals: 5 males and 5 females
Age at treatment: 8 weeks (male); 12 weeks (females)
Identification: By unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: Under laboratory conditions after health examination. Only healthy animals were used for the study.

Conditions
Standard Laboratory Conditions.
Air-conditioned with ranges for room temperature of 22 ± 3 °C and relative humidity 30-70 % and approximately 10-15 air changes per hour. 12 hours artificial fluorescent light/12 hours dark, music during the light period.
Accommodation: During acclimatization in groups of five per sex in Makrolon type-4 cages with standard softwood bedding. Individually in Makrolon type-3 cages with standard softwood bedding during treatment and observation.
Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet (batch 001/06) ad libitum.
Water: Community tap water, ad libitum.

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

The test item was applied at a dose of 2000 mg/kg body weight evenly on the intact skin with a syringe and covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.

Application volume/kg body weight: 6 mL

Duration of exposure:

24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

One day before treatment, the backs of the animals were clipped with an electric clipper, exposing an area of approximately 10 % of the total body surface. Only those animals without injury or irritation on the skin were used in the test. On test day 1, the test item was applied at a dose of 2000 mg/kg body weight evenly on the intact skin with a syringe and covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.
Application volume/kg body weight: 6 mL
Twenty-four hours after the application the dressing was removed and the skin was flushed with lukewarm tap water and dried with disposable paper towels. Thereafter, the reaction sites were assessed.

Statistics:

No statistical analysis was used.

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:

other: No systemic signs of toxicity were observed during the study period. A purple staining of the treated skin was noted in all animals on days 2 and 3 of the study. The staining persisted still on day 4 in one female.

Gross pathology:

No macroscopic findings were observed at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

The median lethal dose of test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat, dermal): greater than 2000 mg/kg body weight

Executive summary:

Five male and five female HanRcc:WIST (SPF) rats were treated with test item at 2000 mg/kg by dermal application. The test item was diluted in vehicle (purified water) at a concentration of 0.33 g/mL and administered at a volume dosage of 6 ml/kg. The application period was 24 hours. 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 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Local signs were noted once daily from test day 2 to 15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. 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. No clinical signs were observed during the course of the study. The treated skin area was stained purple in all animals on days 2 and 3 after treatment. The staining persisted still on day 4 in one female. Otherwise, no dermal symptoms were noted. The body weight of the animals was within the range commonly recorded for this strain and age with the exception of one female, which lost 1.4 % of body weight during the first week after treatment. This animal recovered towards the end of the study. No macroscopic findings were observed at necropsy. Therefore, the LD50 of the test article was estimated to be greater than 2000 mg/kg.bw, which indicates that test article shall not be classified in accordance with CLP (Regulation EC No. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Oral toxicity test and dermal toxicity test of the test substance were performed under GLP conditions according to the OECD guideline. The inhalation toxicity test is waived according to column 2 of REACH Annex VIII.

 

Acute oral toxicity

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with test item 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. All animals survived until the end of the study period. Mild or moderate clinical signs of toxicity (ruffled fur, hunched posture, sedation) were noted in the animals on the day of dosing and a slightly ruffled fur persisted in two animals still on day 2. From day 3 on for the remaining observation period all animals were absent of clinical findings. The body weight of the animals was within the range commonly recorded for this strain and age. No macroscopic findings were recorded at necropsy. Therefore, the LD50 of test item can be estimated to be greater than 2000 mg/kg bw.

 

Acute dermal toxicity

Five male and five female HanRcc:WIST (SPF) rats were treated with test item at 2000 mg/kg by dermal application. The test item was diluted in vehicle (purified water) at a concentration of 0.33 g/mL and administered at a volume dosage of 6 mL/kg. The application period was 24 hours. No deaths occurred during the study. No clinical signs were observed during the course of the study. The treated skin area was stained purple in all animals on days 2 and 3 after treatment. The staining persisted still on day 4 in one female. Otherwise, no dermal symptoms were noted. The body weight of the animals was within the range commonly recorded for this strain and age with the exception of one female, which lost 1.4 % of body weight during the first week after treatment. This animal recovered towards the end of the study. No macroscopic findings were observed at necropsy. Therefore, the LD50 of test article was estimated to be greater than 2000 mg/kg.bw.

 

Acute inhalation toxicity waiver:

Currently no study to assess the acute inhalation toxicity potential of Reactive Brown 051 is available. The calculated value for vapour pressure was found to be 3.6E-37 Pa at 25 °C. Hence, the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Based on column 2, ‘Specific rules for adaptation from column 1’ of the table given in REACH Annex VII, the study on acute inhalation toxicity only needs to be conducted if an exposure via inhalation is to be expected, based on vapour pressure and/or the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the chemical is imported into the EU in a formulated form as a dust-free powder or as a granulate, the exposure via inhalation is considered to be unlikely. Further, the chemical is found to have water solubility of > 306 g/L, hence, in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. The chemical showed low toxicity potential in the available acute oral and dermal toxicity studies (LD50: >2000 mg/kg bw), with no systemic toxicity being seen, hence it does not need to be classified STOT SE and low toxicity is expected for this chemical via the inhalation route. Taking into consideration the above arguments, low toxicity potential is expected on acute exposure of Reactive Brown 051 via inhalation route and hence testing by the inhalation route was considered scientifically not necessary.

Justification for classification or non-classification

Based on the acute oral and dermal toxicity test results with no mortality observed at 2000 mg/kg bw and no specific target organ effects seen, the test substance is not classified according to the CLP Regulation (Regulation EC No. 1272/2008) for acute toxicity or specific target organ toxicity, single exposure.