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EC number: 282-846-2 | CAS number: 84434-47-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication
Data source
Reference
- Reference Type:
- publication
- Title:
- Short-term toxicity study of Green S in rats
- Author:
- S. A. CLODE, I. F. GAUNT, R. J. HENDY*, R. C. COTTRELLt and S. D. GANGOLLI
- Year:
- 1 987
- Bibliographic source:
- Food Chem. Toxic. Vol. 25, No. 12, pp. 969-975, 1987
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- Repeated dose subscute toxicity study was performed to determine the toxic nature of Green S in rats
- GLP compliance:
- not specified
- Limit test:
- no
Test material
- Reference substance name:
- Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- EC Number:
- 221-409-2
- EC Name:
- Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Cas Number:
- 3087-16-9
- Molecular formula:
- C27H26N2O7S2.Na
- IUPAC Name:
- Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Test material form:
- solid
- Details on test material:
- - Name of test material (as cited in study report): Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Molecular formula :C27H26N2O7S2.Na
- Molecular weight: 576.623 g/mol
- Substance type:Organic
- Physical state:Solid
Constituent 1
- Specific details on test material used for the study:
- - Name of the test material: Lissamine Green B
- EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
- Molecular formula: C27H26N2O7S2.Na
- Molecular Weight: 576.623 g/mol
- Substance type: Organic
- Smiles: c12c(\C(c3ccc(N(C)C)cc3)=C3/C=C\C(=[N+](/C)C)C=C3)c(O)c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O-])cc2.[Na+]
- Purity: 82%
- Impurity: volatile matter at 135°C, 3.37%; water-insoluble matter, 0.01%; sodium chloride, 1.4%; sodium sulphate, 8.5%; pH of a 1% solution in distilled water, 5.4; lead, <5 ppm; copper, 4ppm; chromium, 6ppm; zinc, 9 ppm; iron, 30 ppm; cadmium, < 1 ppm; mercury, 0.2ppm; free aromatic amines (as aniline), 29ppm; Miehler's hydrol, <0.01%; Michler's ketone, < 0.01%; R-acid, 0.11%.
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- No data
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Specified-pathogen-free colony (Olac (1976) Ltd, Bieester, Oxon).
- Age at study initiation: Weanling rats
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed 5 per cage in plastic and stainless-steel grid-floored cages suspended on racks over trays lined with paper on which excreta were collected.
- Diet (e.g. ad libitum): Basic diet, Laboratory Diet No. 5 (Spratt's Patent Ltd, Barking, Essex), ad libitum
- Water (e.g. ad libitum): Tap-water, ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2°C
- Humidity (%):40-70 %
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available
IN-LIFE DATES: From: To: No data available
Administration / exposure
- Route of administration:
- oral: feed
- Details on route of administration:
- No data
- Vehicle:
- other: Feed
- Remarks:
- Basic diet, Laboratory Diet No. 5
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Fresh diets were prepared containing Green S at concentrations calculated on the basis of body weight and food intake data to achieve the intended mg/kg/day intakes of 0, 250, 500 or 1000 mg/Kg/day.
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basic diet, Laboratory Diet No. 5
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basic diet, Laboratory Diet No. 5
- Concentration in vehicle: 0, 250, 500 and 1500 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Diets were analysed after preparation and accepted for use if the result was within 10% of the desired value.
- Duration of treatment / exposure:
- 2 weeks (14 days)
- Frequency of treatment:
- Daily
Doses / concentrations
- Remarks:
- 0, 250, 500 and 1500 mg/kg/day
- No. of animals per sex per dose:
- Total: 40
0 mg/kg/day: 5 male, 5 female
250 mg/kg/day: 5 male, 5 female
500 mg/kg/day: 5 male, 5 female
1500 mg/kg/day: 5 male, 5 female - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: No data available
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available - Positive control:
- No data
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: No data available
- Time schedule: No data available
- Cage side observations checked in table [No.?] were included. No data available
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data available
BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of treatment and twice weekly throughout the study.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Measured over the intervals between weighings.
OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes, barbiturate anaesthesia was used.
- Animals fasted: Overnight fasting (with water available)
- How many animals: In control and high dose group
- Parameters checked in table [No.?] were examined: Red blood cells, haemoglobin, methaemoglobin, packed cell volume; Heinz bodies and different type of leucocytes, reticulocytes, neutrophils, eosinophils, lymphocytes and monocytes were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: No data available
- How many animals: All the 40 animals
- Parameters checked in table [No.?] were examined. urea, glucose, total protein and albumin content, glutamic-oxalacetic transaminase, glutamic-pyruvic
transaminase and lactic dehydrogenase were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment urine was collected over a 6-hours period, urine produced in a 6-hr period without water, in a 4- hour period commencing after 16 hours without water and in a 2- hours period immediately after an oral water load of 25 ml/kg body weight.
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined: Specific gravity, Volume, pH, Cell excretion was examined. The renal concentrating and diluting ability were assessed by measuring the volume and specific gravity of urine produced in a 6-hr period without water, in a 4-hr period commencing after 16hr without water and in a 2-hr period immediately after an oral water load of 25 ml/kg body weight. A urinary cell count was made using the latter sample.
NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data available
OTHER:
Organ weight: Yes
Brain, pituitary, heart, stomach, caecum (with and without its contents), kidneys, adrenals, gonads, spleen, thyroid and liver were weighed. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, all macroscopic abnormalities were noted
Organ weight: Yes
Brain, pituitary, heart, stomach, caecum (with and without its contents), kidneys, adrenals, gonads, spleen, thyroid and liver were weighed.
HISTOPATHOLOGY: Yes, Tissue that appeared abnormal was preserved in 10% buffered formalin. Paraffin-wax sections of these tissues from the control rats and those given the highest dietary level were stained with haematoxylin and eosin for histopathological investigation. This investigation indicated the need to examine, at the intermediate dose level, the intestine and thyroids. Hence sections of these were prepared and examined.
Organ and tissue examined:
Brain, pituitary, heart, stomach, caecum (with and without its contents), kidneys, adrenals, gonads, spleen, thyroid and liver skin, eye and optic nerve, Harderian gland, salivary glands, trachea, oesophagus, pancreas, rectum, colon,
urinary bladder, urethra, spinal cord, skeletal muscle, lymph nodes, aorta, vena cava, thymus, lung, prostate, epididymis, seminal vesicles, uterus, vagina and mammary glands and any other tissue that appeared abnormal were examined. - Other examinations:
- No data
- Statistics:
- Statistical analysis of body weight, food intake, water intake, dietary substance intake, haematological examinations and organ weight were performed by using t-test, urine analysis by using (sum of ranks test, (Wilcoxon, 1945) and clinical chemistry by using the exact test of Fisher, 1934.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical signs:
Green staining of fur, extremities and faeces was observed, the intensity of colouring related to the dose. The faeces, particularly those of the 1500 mg/kg/day dose group, were very soft and moist. - Mortality:
- not specified
- Body weight and weight changes:
- not specified
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No significant effects were observed in 1500 mg/kg/day treated rats as compared to control.
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In dilution test, Specific gravity and Cell excretion were significantly decreased and urine volume was increased in 1500 mg/kg/day treated male rats and urine volume was decreased in female rat as compared to control.
Higher levels of urinary protein were observed in 1500 mg/kg/day treated female rats as compared to control. - Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Thyroids weight was decreased in 500 and 1500 mg/kg/day in male rat as compared to control. This was not seen in the females at the same time or in either sex at the laterexaminations.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Green colouring of gastro-intestinal tract were observed in all the treated animals as compared to control.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increases in the numbers of prominent lymph nodes of the gastro-intestinal tract were observed in female rats treated with 1500 mg/kg/day as compared to control.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
Effect levels
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect on Clinical signs, Haematology, Clinical chemistry, Urinanalysis, Gross pathology and histopathology
- Remarks on result:
- other: No toxic effect were observed
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Much of the colour encountered in the urine was due to contamination from these sources. Production of soft, moist faeces and the slightly greater caecal contents were due to an increased osmotic activity of the contents of the gastro-intestinal tract caused by the unabsorbed colouring. The enlarged lymph nodes of the intestine found be the result of a local infection than an effect of the treatment.
Table . Incidence of various reactions for protein in urine samples from rats fed diets to provide intakes of 0 - 1500mg Green S/kg/day for 2 wk
Dose level (mg/kg/day) |
No. of samples giving positive reactions for protein |
|||||||||
Males |
Females |
|||||||||
t |
+ |
++ |
+++ |
++++ |
t |
+ |
++ |
+++ |
++++ |
|
0 |
0 |
0 |
2 |
3 |
0 |
0 |
4 |
1 |
0 |
0 |
250 |
0 |
1 |
0 |
4 |
0 |
0 |
4 |
0 |
1 |
0 |
500 |
0 |
1 |
2 |
2 |
0 |
0 |
3 |
2 |
0 |
0 |
1500 |
0 |
0 |
0 |
2 |
3 |
0 |
0 |
0 |
5* |
0 |
Figures marked with an asterisk are significantly greater (the exact test of Fisher, 1934) than those of the control: *P < 0.05.
Table . Results of renal concentration and dilution tests, urinary pH determinations and urinary cell counts in rats fed diets to provide intakes of 0 -1500mg Green S/kg/day for 2 wk
Dose level (mg/kg/day) |
Concentration test |
Dilution test (24 hrs) |
||||||
Specific gravity |
Volume (mL) |
pH at 0-6 hr |
Specific gravity |
Volume (mL) |
Cell excretion (1000/hr) |
|||
Male |
|
|
|
|
|
|
|
|
0 |
1.050 |
1.101 |
2.3 |
0.2 |
7.8 |
1.031 |
2.6 |
1.5 |
250 |
1.045 |
1.100 |
2.1 |
0.3 |
7.3 |
1.020 |
3.3 |
1.7 |
500 |
1.037 |
1.097 |
2.4 |
0.4 |
7.1 |
1.020 |
3.4 |
0.3* |
1500 |
1.046 |
1.080 |
2.7 |
0.4 |
7.7 |
1.006* |
5.5* |
0.5* |
Female |
|
|
|
|
|
|
|
|
0 |
1.039 |
1.096 |
1.8 |
0.2 |
7.9 |
1.010 |
4.5 |
0.8 |
250 |
1.050 |
1.089 |
1.7 |
0.3 |
7.9 |
1.010 |
4.5 |
0.8 |
500 |
1.046 |
1.086 |
1.2 |
0.2 |
7.3 |
1.016 |
3.0* |
0.7 |
1500 |
1.058* |
1.098 |
1.2 |
0.2 |
7.1 |
1.019 |
3.0 |
1.2 |
Table . Results of haematological examinations of rats fed diets to provide intakes of 0- 1500g Green S/kg/day for 2 wk
Dose level (mg/kg/day) |
RBC (106/mm3) |
Hb (g/100 mL) |
MetHb (% Hb) |
PCV (%) |
Retics (% RBC) |
Leucocytes |
||||
Total |
Differential (%) |
|||||||||
(103/mm3) |
N |
E |
L |
M |
||||||
Male |
|
|
|
|
|
|
|
|
|
|
0 |
7.70 |
16.6 |
0.8 |
42 |
1.4 |
4.4 |
6 |
6 |
90 |
2 |
1500 |
7.62 |
15.5 |
1.7 |
42 |
1.0 |
6.4 |
10 |
0 |
88 |
1 |
Female |
|
|
|
|
|
|
|
|
|
|
0 |
7.65 |
14.7 |
1.6 |
44 |
0.9 |
3.4 |
7 |
1 |
89 |
3 |
1500 |
7.55 |
14.6 |
1.8 |
45 |
1.5 |
3.9 |
14 |
2 |
81 |
3 |
Applicant's summary and conclusion
- Conclusions:
- The No Observed Adverse Effect level (NOAEL) was considered to be 500 mg/kg/day when Wistar male and female rats were treated with Green S for 2 weeks.
- Executive summary:
Repeated dose subscute toxicity study was performed to determine the toxic nature of Green S in rats. Wistar male and female rats were treated with Green S in the concentration of 0, 250, 500 and 1500 mg/kg/day orally in diet for 2 weeks. The animals were observed for clinical signs, mortality, body weight and food intake, hematology, clinical chemistry, urinalysis, gross and histopathological changes. No effects were observed in hematology of treated rats. Green staining of fur, extremities and faeces in all treated rats were observed. Higher levels of urinary protein and in dilution test, Specific gravity and Cell excretion were significantly decreased and urine volume was increased in 1500 mg/kg/day treated male rats and urine volume was decreased in female rat. In addition, Green colouring of gastro-intestinal tract were observed in all the treated animals and Increases in the numbers of prominent lymph nodes of the gastro-intestinal tract were observed in female rats treated with 1500 mg/kg/day as compared to control. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 500 mg/kg/day when Wistar male and female rats were treated with Green S for 2 weeks.
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