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Description of key information

LD50 (rat,oral,female) is greater than 2000 mg/kg bw.
LD50 (rat, dermal, male/female) is greater than 2000 mg/kg bw.

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:
Experimental Starting Date - 18 Apr, 2006;
Experimental Completion Date - 11 May 2006;
Study Completion Date - 13 Jun, 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 (Acute Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010
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 when treated: 12 weeks
Identification: 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 animals without any visible signs of illness were used for the study.

HUSBANDRY:
Conditions Standard Laboratory: Conditions. Air-conditioned with 10 - 15 air changes per hour, and continuously monitored environment with ranges for room temperature 22 ± 3 °C and for relative humidity between 30 - 70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.
Accommodation: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
Diet:Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 001/06 ad libitum.
Water: Community tap water from Füllinsdorf 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 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:
2 groups of 3 females
Control animals:
no
Details on study design:
Two groups, each of three female HanRcc.WIST (SPF) rats, were treated with the 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.
Key result
Sex:
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: Slightly ruffled fur was noted in all animals from the 30-minute or 1-hour reading to the 3-hour reading and persisted in three animals until the 5-hour reading. Soft black feces were observed in the cage of the second treated group at the 5-hour reading
Gross pathology:
No macroscopic findings were recorded at necropsy.
Interpretation of results:
GHS criteria not met
Conclusions:
LD50 (female rat, oral): greater than 2000 mg/kg body weight
Executive summary:

In a GLP-compliant study performed according to OECD test guideline 423, two groups, each of three female HanRcc.WIST (SPF) rats, were treated with the 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.


Slightly ruffled fur was noted in all animals from the 30-minute or 1-hour reading to the 3-hour reading and persisted in three animals until the 5-hour reading. Soft black feces were observed in the cage of the second treated group at the 5-hour reading whereas black feces were observed in the cage of the first treated group on test day 2. 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 the test item after single oral administration to female rats, observed over a period of 14 days is: LD50 (female rat): greater than 2000 mg/kg body weight and it shall not be classified according to the CLP Regulation (Regulation EC No. 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
> 2 000 mg/kg bw
Quality of whole database:
Good quality database with Klimisch rating 1.

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 - 18 April 2006;
Experiment completion date - 09 May 2006;
Study completion date - 13 June 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 (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: RCC Ltd, Laboratory Animal Services CH-4414 Füllinsdorf / Switzerland
Age when treated: Males - 8 weeks, Females - 12 weeks
Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard Laboratory Conditions. Air-conditioned with 10 - 15 air changes per hour, and continuously monitored environment with ranges for room temperature 22 ±3 °C and for relative humidity between 30 - 70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime 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 ("Lignocel", Schill AG, CH-4132 Muttenz) during treatment and observation.
Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 001/06 (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) ad libitum. Results of analyses for contaminants are archived at RCC Ltd.
Water: Community tap water from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd.
Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
On test day 1, the test item was applied evenly at a dose of 2000 mg/kg body weight 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:
Twenty-four hours
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
Five male and five female HanRcc:WIST (SPF) rats were treated with the 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.

NECROPSY: All animals were killed at the end of the observation period by intraperitoneal injection of pentobarbitone at a dose of at least 2.0 mL/kg body weight and discarded after macroscopic examinations were performed. No organs or tissues were retained.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed.
Clinical signs:
other: No systemic signs of toxicity were observed during the study period. Slight black staining of the treated skin area produced by the test item was noted in all animals after removal of the dressing on test day 2 and persisted in one male animal on test day
Gross pathology:
No macroscopic findings were observed at necropsy.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 (rat, dermal) of FAT 40825/A was found to be greater than 2000 mg/kg body weight
Executive summary:

In a GLP-compliant study conducted according to OECD test guideline 402, five male and five female HanRcc:WIST (SPF) rats were treated with the 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 systemic signs of toxicity were observed throughout the whole observation period. Slight black staining of the treated skin area produced by the test item was noted in all animals after removal of the dressing on test day 2 and persisted in one male animal on test day 3. Slight crusts were noted in one female on test day 11 and slight erythema was noted in the same animal on test day 12. 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 the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat): greater than 2000 mg/kg body weight and it shall not be classified according to the CLP Regulation (Regulation EC No. 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
> 2 000 mg/kg bw
Quality of whole database:
Good quality database with Klimisch rating 1.

Additional information

Acute oral toxicity


Two groups, each of three female HanRcc.WIST (SPF) rats, were treated with the 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. Slightly ruffled fur was noted in all animals from the 30-minute or 1-hour reading to the 3-hour reading and persisted in three animals until the 5-hour reading. Soft black faeces were observed in the cage of the second treated group at the 5-hour reading whereas black faeces were observed in the cage of the first treated group on test day 2. 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 the test item after single oral administration to female rats, observed over a period of 14 days is: LD50 (female rat): greater than 2000 mg/kg body weight.


 


Acute dermal toxicity:


Five male and five female HanRcc:WIST (SPF) rats were treated with the 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 systemic signs of toxicity were observed throughout the whole observation period. Slight black staining of the treated skin area produced by the test item was noted in all animals after removal of the dressing on test day 2 and persisted in one male animal on test day 3. Slight crusts were noted in one female on test day 11 and slight erythema was noted in the same animal on test day 12. 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 the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat): greater than 2000 mg/kg body weight.


 


 


Acute inhalation waiver:


Currently no study to assess the acute inhalation toxicity potential of Reactive Green 034 is available. The calculated value for vapour pressure was found to be 1.73 x 10E-32 Pa at 25 °C. Hence, the substance is considered to have low volatility. Synthesis and formulation of this chemical is performed in a closed process; the final product consists of liquid formulations only. 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 the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the substance is imported into the EU in a formulated form as a liquid formulation, the exposure via inhalation is unlikely. Further, Reactive Green 034 was found to be miscible in water (water solubility 341 g/L) and have low log partition coefficient (≤-4.7), 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 (LD50 >2000 mg/kg bw) in the available acute oral and dermal toxicity studies with no mortality or systemic toxicity, hence it does not need to be classified as STOT SE. Taking  the above arguments into consideration, low toxicity potential is expected on acute exposure of Reactive Green 034 via inhalation routes and hence acute toxicity testing by the inhalation route was considered scientifically not necessary.

Justification for classification or non-classification

Based on the results of oral and dermal toxicity tests of the substance, it is not classified as per EU CLP-Regulation (Regulation (EC) No 1272/2008).