[4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate

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:

1987

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

Test material information

Constituent 1

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

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 (10^6/mm^3)	Hb (g/100 mL)	MetHb (% Hb)	PCV (%)	Retics (% RBC)	Leucocytes
						Total	Differential (%)
						(10^3/mm^3)	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.