Trisodium 2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic 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:

LONG-TERM TOXICITY OF SUNSET YELLOW FCF IN MICE

Author:

I. F. GAUNT and P. L. MASON P. GRASSO and IDA S. Kiss

Year:

1974

Bibliographic source:

Food and Cosmetic toxicology, Vol. 12, pp. 1-10, 1974.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

80 weeks chronic repeated dose oral toxicity study of Sunset Yellow FCF was performed to determine its chronic toxic nature

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate

EC Number:

220-491-7

EC Name:

Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate

Cas Number:

2783-94-0

Molecular formula:

C16H12N2O7S2.2Na

IUPAC Name:

disodium 6-hydroxy-5-[(4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate

Details on test material:

- Name of test material: Sunset Yellow FCF
- Molecular formula: C16H12N2O7S2.2Na
- Molecular weight: 454.38 g/mol
- Substance type: organic
- Physical state: solid

Specific details on test material used for the study:

- Name of test material: Sunset Yellow FCF
- IUPAC name: disodium 6-hydroxy-5-[(4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate
- Molecular formula: C16H12N2O7S2.2Na
- Molecular weight: 454.38 g/mol
- Substance type: organic
- Physical state: solid
- Impurity: Dye content*, min. 85%; subsidiary dyes*, max 3%; matter volatile at 135°C *, max 10%; matter insoluble in water*, max 0.1%; matter soluble in diisopropyl ether*, max 0.2%; chloride and sulphate (as sodium salts)*, max 5.0%; copper*, max 10 ppm; arsenic, max 1 ppm; lead*, max l0 ppm; heavy metals (as sulphides)*, not producing more intense colour than reference standard.

Test animals

Species:

mouse

Strain:

other: Charles River CD

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding colony
- Weight at study initiation: Male- 21-30 g and female - 17-25 g
- Fasting period before study: no
- Housing: housed in cages of 15
- Diet (e.g. ad libitum): ground Oxoid pasteurized breeding diet; ad libitum
- Water (e.g. ad libitum): water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 1°C
- Humidity (%):50-60%

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: basic diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose levels of 0, 100, 200, 400, 800 mg/l (0, 0.2, 0.4, 0.8 or 1.6%)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): Oxoid pasteurized breeding diet
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Oxoid pasteurized breeding diet
- Concentration in vehicle: 0, 100, 200, 400, 800 mg/l (0, 0.2, 0.4, 0.8 or 1.6%)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 200, 400, 800 mg/l (0, 0.2, 0.4, 0.8 or 1.6%)
Basis:
nominal in diet

No. of animals per sex per dose:

Control group:60 male and 60 females
Test group:
100 mg/Kg: 30 male and 30 females
200 mg/Kg: 30 male and 30 females
400 mg/Kg: 30 male and 30 females
800 mg/Kg: 30 male and 30 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Continued surveillance
- Cage side observations checked in table [No.?] were included. Any abnormalities in condition and behavior

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: At 4 weeks interval throughout the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: wk 13, 26 and 52 and from all surviving mice at wk 80
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10/sex from control group and animals fed 400 and 800 mg/Kg
- Parameters checked in table [No.?] were examined. Haemoglobin concentration and packed cell volume and counts were made of erythrocytes and total leucocytes. Differential leucocyte counts and reticulocyte counts were made on the blood from the mice fed on the control diet and from those given diet containing 1.6% Sunset Yellow FCF at wk 26, 52 and 80, and reticulocyte counts were also carried out for these groups at wk 13.

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, At autopsy, all macroscopic abnormalities were noted and the brain, heart, liver, kidneys, spleen, stomach, small intestine, caecum and testes were weighed.

HISTOPATHOLOGY: Yes, the brain, heart, liver, kidneys, spleen, stomach, small intestine, caecum and testes together with salivary gland, thyroid, adrenals, lymph
nodes, pancreas, pituitary, ovaries, uterus, urinary bladder, lungs, colon, rectum, spinal cord, skeletal muscle and any other tissue that appeared abnormal were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haemotoxylin and eosin. All tissues from the control mice and those fed 800 mg/Kg Sunset Yellow FCF were examined microscopically, while at the lower dietary levels examination was confined to the liver and kidney together with any tissue seen to be abnormal at autopsy.

Other examinations:

Behavior and were weighed at 4-wk intervals throughout the study. During the first half of the study it was noticed that there was a tendency for the male mice to fight. Bite lesions of the anogenital region were particularly frequent and these were associated with obstructions of the urinary tract. To avoid further fighting, all the mice were caged individually from month 8.

Statistics:

No data

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

There were deaths in all groups during the study and there was no relationship between the number of deaths at any time and the dietary intake of Sunset Yellow FCF

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The terminal body weights and the body-weight gains were similar in all groups.

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 evidence of any haematological adverse effect due to the administration of Sunset Yellow FCF.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences between the organ weights or relative organ weights of the test groups and the corresponding controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Distension of the bladder, frequently associated with hydroureter and hydronephosis was noted. Pus and hard protein deposits were found in the bladders of many of the mice and in a number of cases it was possible to show that an obstruction had occurred in the urethra in the region of the bulbo-urethral complex. After the male mice were put into individual cages, there was a marked reduction in the number showing this syndrome, although isolated cases of urinary retention were seen throughout the study.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No differences between treated and control mice in the incidence or severity of the lesions seen. There as an unusually high incidence of pyelonephritis and chronic inflammation of the bladder and urethra, but these lesions occurred mainly in the male mice killed because of urinary retention in the early stages of the study.

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

Of the malignant tumours found, lymphomas and reticulum-cell neoplasms occurred in treated and control mice. In addition a splenic haemangiosarcoma and a mammary carcinoma were found in the female mice fed on the diet containing the lowest level of Sunset Yellow FCF (100 mg/Kg). A single tumour of the Harderian gland was found in a female from the group given the 1.6% level. The commonest benign tumours were pulmonary adenomas and benign cysts in the ovary, found with a similar frequency in treated and control mice. The only other tumour occurring in treated mice without a similar finding in the controls was a granulosa-cell tumour of the ovary in a mouse given 800 mg/Kg Sunset Yellow FCF.

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

800 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: neoplastic

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: No significant alterations were noted between the treated and control animals

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Cumulative death record of mice fed Sunset Yellow FCF at dietary levels of 0-1.6 % for 80 wk

Wk no.	Cumulative mortality
	Males					Females
	0	0.2	0.4	0.8	1.6	0	0.2	0.4	0.8	1.6
8	0	0	0	0	0	0	0	0	0	1
16	0	1	0	1	1	1	0	0	0	1
24	5	6	2	5	2	1	0	0	0	1
32	7	10	6	8	6	2	0	0	0	2
40	9	12	7	9	9	6	0	1	1	2
48	13	12	9	9	10	11	0	1	2	6
56	16	16	9	12	10	15	1	3	3	7
64	19	16	9	13	11	16	2	5	5	7
72	23	18	14	14	16	21	7	9	5	9
80	27	21	17	16	19	26	10	12	7	14

 

Table 2. Mean body weights of mice fed on diets containing 0-1.6% Sunset Yellow FCF for 80 wk

Dietary level (%)	Body weight (g) at week						Weight gain (g) at wk 81
	0	13	33	49	65	81
Males
0	35.8	46.7	48.5	48.5	51.7	49.4	13.6
0.2	26.4	46.6	48.9	48.6	50.1	48.6	12.2
0.4	36.0	46.8	49.2	48.7	51.5	50.1	14.1
0.8	35.7	46.0	48.0	46.4	50.8	48.5	12.8
1.6	35.6	47.2	50.2	49.8	51.0	49.4	13.8
Females
0	27.9	42.5	49.4	45.0	48.3	45.1	17.2
0.2	28.1	44.0	52.8	46.0	50.4	49.8	21.7
0.4	28.3	41.5	50.1	43.9	49.6	45.9	17.6
0.8	27.7	42.1	50.8	44.2	50.0	48.7	21.0
1.6	27.5	41.7	49.1	44.9	51.5	48.0	20.5

 

Table 3. Relative organ weights of mice fed diets containing 0-1.6% Sunset Yellow FCF for 80 wk

Sex and dose	No. of mice examined	Relative organ weights (g/100 g bw)									Terminal bw (g)
		Brain	Heart	Liver	Spleen	Kidneys	Stomach	Small intestine	Caecum	Testes
Male
0	28	1.15	0.64	6.66	0.51	1.83	0.91	4.47	0.60	0.57	47
0.2	8	1.22	0.62	6.22	0.49	1.89	1.07	4.67	0.71	0.69	45
0.4	11	1.23	0.64	6.09	0.48	2.05	0.95	4.36	0.68	0.68	44
0.8	12	1.13	0.64	6.49	0.58	1.96	1.00	4.69	0.73	0.60	45
1.6	10	1.17	0.70	6.87	0.59	1.89	0.96	4.96	0.65	0.63	46
Females
0	25	1.41	0.56	5.49	0.90	1.41	1.18	4.26	0.67	-	39
0.2	18	1.26	0.52	5.48	0.79	1.40	1.00	4.19	0.62	-	42
0.4	15	1.38	0.56	6.51	0.83	1.44	1.07	5.11	0.74	-	39
0.8	21	1.29	0.55	5.29	0.69	1.40	1.02	4.00	0.64	-	42
1.6	12	1.32	0.56	6.34	0.98	1.41	1.05	3.72	0.63	-	41

 

Table 4. Results of haematological examinations in mice fed on diets containing 0-1.6% Sunset Yellow FCF for 80 wk

Sex and dose	No. of mice examined	Hb (g/100mL)	Pvc (%)	Rbc (10^6/mm^3)	Retics (% of RBC)	Leucocytes
						Total (10^3/mm^3)	Differential
							N	E	L	M
	Week 52
Males
0	10	12.5	47	8.18	3.7	17.07	22	0	72	6
0.8	10	11.9	45	4.84	-	11.68	-	-	-	-
1.6	10	11.0	44	7.65	2.3	10.63	16	0	82	2
Females
0	10	14.2	47	8.11	3.2	6.59	20	0	78	2
0.8	10	12.6	47	8.32	-	8.60	-	-	-	-
1.6	10	11.0	44	7.54	2.3	7.61	23	0	76	1
	Week 80
Males
0	31	12..4	39	6.69	5.5	5.60	44	0	54	2
0.2	9	13.2	37	6.62	-	5.19	-	-	-	-
0	13	11.9	38	6.77	-	4.29	-	-	-	-
0.8	12	12.2	38	6.49	-	5.33	-	-	-	-
1.6	10	13.5	40	6.96	6.2	6.25	52	0	45	3
Females
0	31	12.7	40	6.95	5.8	6.02	61	0	31	8
0.2	19	12.9	41	6.96	-	4.97	-	-	-	-
0	18	12.5	39	6.70	-	6.00	-	-	-	-
0.8	23	12.7	40	6.74	-	5.97	-	-	-	-
1.6	12	12.6	40	6.47	6.3	5.63	64	0	33	3

 

Table 5. Incidence of histopathological abnormalities (excluding tumaurs) found in mice fed diets containing 0-1"6% Sunset Yellow FCF for 80 wk

Organ and histological finding	No. of mice affected
	Male					Female
	0	0.2	0.4	0.8	1.6	0	0.2	0.4	0.8	1.6
	50	26	30	27	1.9	47	29	27	27	29
Lung
Chronic inflammatory infiltration	3	0	0	0	0	0	1	0	0	1
Liver
Amyloidosis	17	7	7	6	5	5	5	7	7	4
Fatty degeneration	7	1	4	2	0	1	0	0	0	0
Spleen
Amyloidosis	11	10	5	4	2	0	0	0	0	0
Kidney
Degenerative changes	13	4	3	0	1	5	3	3	1	4
Pyelonephritis	4	6	2	3	2	0	0	0	0	0
Bladder
Chronic inflammation	3	5	4	2	1	0	0	0	0	0
Urethra
Chronic inflammation	0	2	1	0	0	0	0	0	0	0
Heart
Interstitial fibrosis	0	0	0	1	0	0	0	0	0	0
Non-specific
Lymphoid hyperplasia	2	0	0	0	0	12	0	3	1	4
Chronic subcutaneous
abscess	0	0	0	0	0	1	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

The 80 weeks chronic repeated dose study on male and female Charles River CD mouse indicated that no effects observed on body weight, organ weight, haematology and histopathology. Thus, on the basis of study results the NOAEL (no observed adverse effect level) was considered to be 800 mg/kg diet.

Executive summary:

Repeated oral toxicity of Sunset Yellow FCF was determined by performing a 80 weeks repeated dose toxicity study usiing Charles River CD mouse (male and female) at dose levels of 0, 100, 200, 400 or 800 mg/Kg (0.2, 0.4, 0.8 or 1.6% respectively) by oral diet route. The animals were observed for clinical signs, mortality, body weight changes, hematology and were subjected to gross and histopathology. The feeding of Sunset Yellow FCF did not adversely affect the death rate within the groups, the rate of body-weight gain, the organ weights or the haematological findings. The incidence and severity of the histopathological findings were similar in treated and control mice and there was no evidence of an increased incidence of tumours in the mice given Sunset Yellow FCF. Distension of the bladder, frequently associated with hydroureter and hydronephosis was noted. Pus and hard protein deposits were found in the bladders of many of the mice and in a number of cases it was possible to show that an obstruction had occurred in the urethra in the region of the bulbo-urethral complex. After the male mice were put into individual cages, there was a marked reduction in the number showing this syndrome, although isolated cases of urinary retention were seen throughout the study. Of the malignant tumours found, lymphomas and reticulum-cell neoplasms occurred in treated and control mice. In addition a splenic haemangiosarcoma and a mammary carcinoma were found in the female mice fed on the diet containing the lowest level of Sunset Yellow FCF (100 mg/Kg). A single tumour of the Harderian gland was found in a female from the group given the 1.6% level. The commonest benign tumours were pulmonary adenomas and benign cysts in the ovary, found with a similar frequency in treated and control mice. The only other tumour occurring in treated mice without a similar finding in the controls was a granulosa-cell tumour of the ovary in a mouse given 800 mg/Kg Sunset Yellow FCF. Thus, on the basis of above results the NOAEL (no observed adverse effect level) for Sunset Yellow FCF was considered to be 800 mg/kg diet.