Disodium 8-hydroxynaphthalene-1,6-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: data from peer - reviewed journals

Justification for type of information:

Data is from publication

Data source

Reference

Reference Type:

publication

Title:

LONG-TERM TOXICITY STUDY OF AMARANTH IN RATS USING ANIMALS EXPOSED IN UTERO

Author:

S. A. CLODE, J. HOOSON, D. GRANT and W. H. BUTLER

Year:

1987

Bibliographic source:

Fd Chem. Toxic. Vol. 25, No. 12, pp. 937-946, 1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

Combined repeated dose & carcinogenicity study of amaranth in rats was conducted to evaluate its toxic nature.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

EC Number:

213-022-2

EC Name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Cas Number:

915-67-3

Molecular formula:

C20H14N2O10S3.3Na

IUPAC Name:

trisodium 3-hydroxy-4-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Amaranth

IUPAC Name:

Amaranth

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Amaranth dye
- Molecular formula : C20H11N2O10S3.3Na
- Molecular weight : 604.4789 g/mole
- Substance type: Organic
- Physical state: Powder
- Impurities (identity and concentrations): 0.9%*; naphthionic acid, 0.05%*; Schaeffer acid, <0.01%*; Racid, 0.06%*; G-acid, <0.01%*; 2-napthol-3,6,8- trisulphonic acid, 0.01"; water-soluble matter, 0.02%*; ether-extractable matter, 0.05%*; primary aromatic amines, < 10ppm*; arsenic, < 1 ppm*; lead, 5 ppm*; copper, 2ppm*; zinc, 5ppm*; antimony, < 1 ppm*; chromium, 5 ppm*; mercury and < 0.5 ppm*

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amaranth dye
- Molecular formula : C20H11N2O10S3.3Na
- Molecular weight : 604.4789 g/mole
- Substance type: Organic

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Specified-pathogen-free breeding colony (Olac (1976) Ltd, Bicester, Oxon)
- Age at study initiation: No data available
- Weight at study initiation: 88 to 91 g male, 79 to 83 g female
- Fasting period before study: No data available
- Housing: Animals were housed in stainless steel and polypropylene grid-floored cages at all stages of the study apart from females in the latter stages of pregnancy and lactation where solid-floored cages were used.
- Diet (e.g. ad libitum): Spratt's Laboratory
Animal Diet No. 2 (Spratt's Patent, Barking, Essex), ad libitum
- Water (e.g. ad libitum): Tap-water, ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 50-70%
- Air changes (per hr): 15-20 air changes/hr with no recirculation.
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Spratt's Laboratory Animal Diet No. 2

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Animals were fed diets containing amaranth at concentrations calculated to provide daily intakes of 0 (control), 50, 250 or 1250 mg/kg body weight.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were adjusted at intervals to maintain a constant daily dosage.
- Mixing appropriate amounts with (Type of food): Spratt's Laboratory Animal Diet No. 2
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Spratt's Laboratory Animal Diet No. 2
- Concentration in vehicle: 0, 50, 250 or 1250 mg/kg body weight.
- 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:

not specified

Duration of treatment / exposure:

111 week for male and 112 week for female

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250 and 1250 mg/kg body weight/day
Basis:

No. of animals per sex per dose:

Total: 624
0 mg/kg bw /day: 114 male, 114 female
50 mg/kg bw /day: 66 male, 66 female
250 mg/kg bw /day: 66 male, 66 female
1250 mg/kg bw /day: 66 male, 66 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included.: Survival were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 17 week and then at 2-week intervals for the remainder of 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: Twice-weekly

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 3 and 6 months from control and 1250 mg/kg bw/day, from all groups at 12 and 18 months and at the study termination
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: From 20 male and 20 female at 3 and 6 months from control and 1250 mg/kg bw/day and from 20 male and 20 female of all treated rats at 12 and 18 months.
- Parameters checked in table [No.?] were examined: Haemoglobin concentration, packed-cell volume, methaemoglobin concentration, total erythrocyte and leucocyte counts were examined.

Reticulocyte and differential leucocyte
counts were examined in 0 and 1250 mg/kg bw/day dose group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination of study
- Animals fasted: No data available
- How many animals: All treated animals were examined.
- Parameters checked in table [No.?] were examined: Content of urea, glucose, albumin and total protein and for the activities of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, lactic dehydrogenase and alkaline phosphatase were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6 and 9 months from 0 and 1250 mg/kg bw/day dose group and at 12, 18 and 24 months from all treated groups.
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined: Appearance, microscopic constituents, semiquantitative tests for the content of protein, glucose, ketones, bile salts and blood, refractive index, volume of urine and number of cells were examined.

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: Brain, Heart, Liver, Spleen, Kidneys, Stomach, Full and Empty Caecum, Adrenals and Gonads were weighted.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross abnormalities were examined at termination of study.

Organs were preserved in 10% buffered formalin.

Paraffin-wax sections of all tissues, except nasal bones and spinal cord, from all treatment groups were stained with haematoxylin and eosin and examined by light microscopy. Sections of the nasal bones and spinal cord were prepared in the same manner from just the control animals and those on the highest dietary level.

HISTOPATHOLOGY: Yes
Organ examined:
Adrenal glands, brain, caecum (with and without its contents), gonads, heart, kidneys, liver, spleen, stomach and thyroid were weighed. Samples of these tissues together with the aorta, urinary bladder, colon, epididymis, eye, Harderian gland, lungs, lymph nodes, mammary gland, skeletal muscle, nasal bones, nerve, oesophagus, pancreas, prostate, salivary glands, seminal vesicles, skin, small intestine, spinal cord, thymus, tongue, trachea, uterus, vagina and vena cava were examined.

Other examinations:

Viability of pups per litter at day 18 was examined.

Statistics:

Statistical analysis were performed by using log-rank analysis for mortality data; Student's t test for body weight, food consumption of F0, water consumption of F0,haematology, urine data, serum analysis, organ weight and relative organ weight data; chi-square test for fertility data; analysis of variance for food consumption of FI, water consumption and pup observation data; test for positive trend using 4 x 2 contingency tables for histology data, tumour incidence and total number of tumour-bearing animals.
These latter data were analysed as recommended by Peto et al. (1980) for a scheduled kill. Neither the time at which findings occurred nor their contribution to the cause of death have been taken into account. Where a positive trend was identified, the findings in each treated group were compared with those of the control group.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

When treated wtih 1260 mg/kg bw/day, related pink coloration of the fur and faeces moist faeces were observed in treated rats as compared to control.

Dose-related pink coloration of the fur and faeces were observed in treated rats due to contamination with test substance

Mortality:

no mortality observed

Description (incidence):

No significant effect on survival of treated rats were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, significant decrease in body weight was observed in male rats from week 4 to 73 and in female from 51 and 73 as compared to control.

Slight effect on body weight was due to a reduction in the absorption or utilization of the nutrient.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, increase in food consumption was observed in treated rats were observed as compared to control.

Due to a reduction in the absorption or utilization of the nutrient.
Compound intake:
Average compound intake of male rats were 47, 242 and 1260 mg/kg bw/day and for female rats 49, 246 and 1260 mg/kg bw/day

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, increase in water consumption was observed in treated male rats as compared to control.

Increased water loss was observed due to production of softer, moister faeces and a compensatory increase in water intake.

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, decreased in packed cell volume was observed at month 6 and 12 months in male and at month 18 in female rats as compared to control.

Statistically significant slightly increased haemoglobin concentrations were observed in treated female rats at termination of study as compared to control.

Observed effect were not dose related or consistent between the sexes

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, decrease in glutamic-oxalacetic transaminase activities was observed in male and female rats but not significant in female rats.

Urinalysis findings:

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, Increase level of protein in urine at 12 months in female rats was observed as compared to control.

Semi-quantitative analysis of urine for bilirubin and ketones was hindered at months 18 and 24 due to the contamination of the urine with amaranth which interfered with the colour reaction.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

When treated with 1260 mg/kg bw/day, statistically significant increase in absolute and relative full and empty caecum weight were observed in treated male and female rats as compared to control.

When treated with 242 mg/kg bw/day, increase in absolute and relative full caecum weight was observed in treated male rats as compared to control.

The observed effect was not statistically significant as compared to control.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Transitional-cell hyperplasia of the bladder, inflammatory cell infiltrate of seminal vesicles and testicular interstitial-cell hyperplasia were observed in 47 mg/kg bw/day male and 1260 mg/kg bw/day treated male and female rats.

Statistically significant increase in renal calcification and renal pelvic epithelial hyperplasia, lung oedema and haemorrhage, of lymph-node haemorrhage and degenerative changes in brain and nerve, egenerative and inflammatory changes in heart, inflammatory changes of the thymus, aortic calcification and atrial thrombi were observed in 1260 mg/kg bw/day treated female rats.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Statistically significant increase in uterine polyps and vaginal fibromas were observed in 1260 mg/kg bw/day treated female rats.

The observed effects were typical of this strain and occur in ageing rats.

Other effects:

no effects observed

Description (incidence and severity):

No effect on No. of litters, number of pups per litter at day 18 and pup weight at day 18 were observed in treated rats as compared to control.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 260 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect on survival, clinical sign, body weight, food consumption, food intake, water consumption, haematology, clinical chemistry, urinalysis, organ weight, gross pathology and histopathology

Remarks on result:

other: No toxic effect observed.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mean body weights of rats fed amaranth at dose levels of 0-1250mg/kg/day

		Mean body weight (g)
		Males				Females
Wk on test	Dose level (mg/kg/day)...	0	50	250	1250	0	50	250	1250
0		91	88	89	87	83	81	79*	80
4		278	274	278	270*	186	185	188	189
8		377	378	382	363**	228	229	233	234*
12		432	432	439	414"*	255	253	258	258
25		515	514	520	488***	286	286	290	283
51		593	600	599	558***	352	346	351	327***
73		603	617	605	564***	390	394	391	354***
97		562	588	576	546	380	399	375	358
109		543	538	548	509	370	386	351	337*

The results are expressed as the means for all survivors in each group. Those marked with asterisks differ significantly (Student's t test) from the control value: *P < 0.05; **P < 0.01; ***P < 0.001.

Mean food and water consumption and calculated intake of colouring by rats fed amaranth in the diet at dose levels of 0-1250 mg/kg/day

Nominal dose level (mg/kg/day)	Food intake (g/rat/day)	Water intake (ml/rat/day)	Calculated intake of amaranth (mg/kg/day)
Males
0	20.5	32.4	--
50	20.4	31.4	47
250	20.8	33.2	242
1250	21.8"**	36.3***	1260
Females
0	17.4	33.5	--
50	17.4	33.4	49
250	32.0	32.0	246
1250	18.1"**	36.9	1260

The figures marked with asterisks differ significantly (Student's t test) from that of the controls: ***P < 0.001.

Organ weights and relative organ weights of rats fed amaranth in the diet at dose levels of 0—1250 mg/kg/day for 2 yr

Dose level (mg/kg/day)	No. of rats examined	Absolute and relative organ weights												Terminal body weight (g)
								Caecum
		Brain	Heart	Liver	Spleen	Kidneys	Stomach	Full	Empty		Adrenalst		Gonads$
		Organ weight (g)
Males
0	19	2.35	1.15	13.99	1.58	4.22	2.29	2.82	1.10		66.3		3.21	522
50	21	2.29	1.44	13.13	1.35*	3.75	2.39	2.78	1.10		70.0		3.28	501
250	16	2.37	1.47	13.58	1.64	3.69	2.20	4.09**	1.20		78.1		2.98	500
1250	18	2.34	1.47	12.92*	1.45	3.79	2.20	8.98***	1.73***		74.1		3.20	468*
Females
0	26	2.17	1.17	10.54	1.13	2.98	1.82	2.71	0.95		49.0		82.5	350
50	20	2.12	1.15	11.04	1.03	2.80	1.84	2.70	0.93		53.4		81.1	361
250	20	2.12	1.10	10.62	1.03	2.76	1.78	2.77	0.96		49.4		72.0	330
1250	13	2.12	1.11	9.85	0.99	2.59	1.61	6.76***	1.31***		55.5		83.3	325
		Relative organ weight (g/100 g bedy weight)
Males
0	19	0.46	0.29	2.72	0.31	0.83	0.45	0.55	0.21	12.9		0.62
50	21	0.46	0.29	2.62	0.27	0.75	0.48	0.55	0.22	14.2		0.70
250	16	0.46	0.30	2.75	0.33	0.75	0.45	0.84**	0.25	15.8		0.60
1250	18	0.51*	0.32	2.81	0.31	0.84	0.48	1.91***	0.37***	16.8		0.70
Females
0	26	0.63	0.34	3.05	0.33	0.88	0.53	0.80	0.28	14.4		23.8
50	20	0.60	0.33	3.12	0.29	0.81	0.53	0.78	0.27	15.1		22.6
250	20	0.65	0.34	3.25	0.32	0.86	0.55	0.87	0.30	15.1		21.8
1250	13	0.66	0.35	3.06	0.31	0.81	0.50	2.07***	0.41***	17.3		25.8

ɫ Absolute and relative weights of this organ are expressed in mg and mg/100 g body weight, respectively.

‡ Absolute and relative weights of female gonads are expressed in mg and mg/100 g body weight, respectively.

Values are the results expressed as mans for the number of rats shown and those marked with asterisks differ significantly the control values: *P < 0.05; **P < 0.01; ***P < 0.001.

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1260 mg/kg body weight /day when Wistar male and female rats were treated with Amaranth dye(915-67-3).

Executive summary:

In acombined repeated dose & carcinogenicity,Wistar male and female rats were treated withAmaranth dye in the concentration of 47, 242 and 1260 mg/kg bw/day for male and 49, 246 and 1260 mg/kg bw/day for female orally in fed.No significant effect on survival of treated rats were observed as compared to control.Significant decrease in body weight in male rats from week 4 to 73 and in female rat from 51 and 73 and increase in food consumption was observed in 1260 mg/kg bw/day treated rats as compared to control. Slight effect on body weight and food consumption was due to a reduction in the absorption or utilization of the nutrient. Average compound intake of male rats were 47, 242 and 1260 mg/kg bw/day and for female rats 49, 246 and 1260 mg/kg bw/day.Increase in water consumption was observed in 1260 mg/kg bw/day treated male rats as compared to control. Increased water loss was observed due to production of softer, moister faeces and a compensatory increase in water intake. Similarly, decreased in packed cell volume was observed at month 6 and 12 in male and at month 18 in female rats and slightly increased haemoglobin concentrations in female rats at termination of study and decrease in glutamic-oxalacetic transaminase activities in male and female rats but not significant in female rats were observed at 1260 mg/kg bw/day. Observed effect were not dose related or consistent between the sexes. Increase level of proteinin urine at 12 months in female was observed at 1260 mg/kg bw/day andSemi-quantitative analysis of urine for bilirubin and ketones was hindered at months 18 and 24 due to the contamination of the urine with amaranth which interfered with the colour reaction. Statistically significant increase in absolute and relative full and empty caecum weight were observed in male and female at 1260mg/kg bw/day andincrease in absolute and relative full caecum weight in male rats at 242mg/kg bw/day were observedas compared to control. The observed effect was not statistically significant as compared to control. In addition, Non-neoplastic transitional-cell hyperplasia of bladder, inflammatory cell infiltrate of the seminal vesicles and testicular interstitial-cell hyperplasia were observed in 47 mg/kg bw/day male and 1260 mg/kg bw/day treated male and female rats. Statistically significant increase in renal calcification and renal pelvic epithelial hyperplasia, lung oedema and haemorrhage, lymph-node haemorrhage and degenerative changes in the brain and nerve, degenerative and inflammatory changes in heart, inflammatory changes in thymus, aortic calcification and atrial thrombi were observed in 1260 mg/kg bw/day treated female rats. Statistically significant increase in neoplastic uterine polyps and vaginal fibromas were observed in 1260 mg/kg bw/day treated female rats. The observed effects were typical of this strain and occur in ageing rats. No effect on number of litters, number of pups per litter at day 18 and pup weight at day 18 were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1260 mg/kg body weight /day when Wistar male and female rats were treated with Amaranth dye orally in fed for 2 years.