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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: data from peer - reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Immunological studies on Amaranth, Sunset Yellow and Curcumin as food colouring agents in albino rats

Author:

Mohamed M. Hashem a, Attia H. Atta, Mahmoud S. Arbid , Somaia A. Nada, Gihan Farag Asaad

Year:

2010

Bibliographic source:

Food and Chemical Toxicology 48 (2010) 1581–1586

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Repeated dose oral toxicity study of Amaranth in rats

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 (E123)
- Molecular formula (if other than submission substance): C20-H11-N2-O10-S3.3Na
C20-H14-N2-O10-S3.3Na
- Molecular weight (if other than submission substance): 604.4789 g/mole
- Substance type: Organic
- Physical state: Powder
- Impurities (identity and concentrations): No data available

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Research Center (Giza, Egypt)
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed 10 per in steel mesh cages
- Diet (e.g. ad libitum): Commercial standard pellets fed enriched with barley and carrots, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- 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: unspecified

Vehicle:

not specified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: No data available

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data available
- Concentration in vehicle: 47 mg/kg bw/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:

not specified

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 and 47 mg/kg bw/day
Basis:

No. of animals per sex per dose:

Total : 20
0 mg/kg bw/day: 10 female
47 mg/kg bw/day: 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Rats administered Amaranth 7.5 mg/kg b. wt. calculated for human and modified according to Paget and Barnes (1964) to be 47 mg/kg b. wt. throughout the experimental period.
- Rationale for animal assignment (if not random): Animals were assigned randomly to test group.
- 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

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: No data available
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: On day 14 and 28 day

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

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): No data available
- Time schedule for examinations: No data available

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: On day 14 and 28 day
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: All 20 animals were tested.
- Parameters checked in table [No.?] were examined. TLC and Differential leucocytic count (Lymphocytes, Neutrophiles, Monocytes, Basophiles and Eosinophiles) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 14 and 28 day
- Animals fasted: No data available
- How many animals: All 20 animals were tested.
- Parameters checked in table [No.?] were examined. Mononuclear cell count, Total serum protein, Albumin, Globulins and A/G ratio were examined.

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

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: Spleen and Thymus gland were weighted.

Sacrifice and pathology:

GROSS PATHOLOGY: No data available

HISTOPATHOLOGY: No data available

Other examinations:

Delayed type hypersensitivity response and Serum protein fractionation were examined

Statistics:

Statistical analyses were performed by using one way ANOVA and SPSS version (9.0). Data were represented as mean ± SE at p ≤ 0.05

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

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

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Details on results:

Mortality: No data available

Clinical signs: No data available

Body weight and weight gain: No significant effect on body weight gain of treated rats was observed as compared to control.

Food consumption and compound intake: No data available

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination No data available

Haematology No significant effects on total leucocytes count were observed in treated rats however, a significant increase in lymphocyte and basophiles % and neutrophils and monocytes % were significantly decreased as compared to control.

Clinical chemistry: Increased number of circulating mononuclear cells in peripheral blood but no effect on Total serum protein, albumin and globulin concentration were observed in treated rats as compared to control.

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights No significant effect on relative Spleen and Thymus gland weight were observed in treated rats as compared to control.

Gross pathology: No data available

Histopathology: No data available

Details on results: Decrease in neutrophiles and monocytes percent could affect the integrity of the cellular immune response.

Delayed hyper sensitivity:
Significantly decreased in oedema % of the paw were observed in treated rats as compared to control.

Serum protein fractionation:
Increases in density of albumin band and no effect on density of globulin region were observed in treated rats as compared to control.

Effect levels

Dose descriptor:

NOAEL

Effect level:

47 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effect on body weight, hematology, clinical chemistry, Organ weight and humoral immune response

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Effect of Amaranth in daily dose of 47 mg/kg b. wt., respectively, for 2 weeks before and 2 weeks after sensitization with sheep RBC’s on body weight and relative organ (mean ± SE, n = 10).

Groups	Body weight (g)	Spleen (% of body weight)	Thymus gland (% of body weight)
Control	216.0 ± 1.71	0.45 ± 0.04	0.22 ± 0.02
Amaranth	229.4 ± 14. 8	8 0.46 ± 0.04	0.18 ± 0.01

* Significant at p 6 0.05compared to control.

Effect of Amaranth in daily dose of 47 mg/kg b. wt., respectively, for 2 weeks before and 2 weeks after sensitization with sheep RBC’s on the total (TLC) and differential leucocytic (DLC) count in albino rats (mean ± SE, n = 10).

Groups	TLC(10^3/mm^3)	Differential leucocytic count (%)
Control	35.84 ± 1.5	61.7 ± 3.51	35 ± 1.73	3.3 ± 1.04	1 ± 0.12	1.2 ± 0.15
Amaranth	32.03 ± 2.1	68.72±3.07*	26.33 ± 2.52*	2.5 ± 0.85*	1.3 ± 0.09*	1.25 ± 0.09

* Significant p 6 0.05 compared to control.

Effect of Amaranth in daily dose of 47 mg/kg b. wt., respectively, for 14 days before and 2 weeks after sensitization with sheep RBC’s on number of circulating mononuclear cells from peripheral blood samples in albino rats (mean ± SE, n = 10).

Groups	Mononuclear cell count X10^3/mm^3
Control	19.77 ± 1.21
Amaranth	24.74 ± 1.19***

* Significant p 6 0.05 compared with control.

** Significant increase.

*** Significant decrease.

Effect of Amaranth in daily dose 47 mg/ kg b. wt. for 4 weeks on delayed hypersensitivity reaction induced by intradermal injection of 0.025 X 10⁹SRBC/ml into right hind foot pad in albino rats in the 30^thday (mean ± SE, n = 10).

Groups	Paw volume		% of oedema
	Before injection	After injection
Control	0.86 ± 0.06	33.75 ± 2.81	33.75 ± 2.81
Amaranth	0.91 ± 0.03	1.25 ± 0.05	26.68 ± 2.29*

* Significant p 6 0.05 compared with control.

Effect of Amaranth in daily doses of 47 mg/kg b. wt., respectively, for 2 weeks before and 2 weeks after sensitization with sheep RBC’s on total serum protein, albumin and globulin (g%) and albumin/ globulin ratio in albino rats (mean ± SE, n = 10).

Groups	Total serum protein (g/dl)	Albumin (g/dl)	Globulins (g/dl)	A/G ratio
Control	6.89 ± 0.2	4.17 ± 0.2	2.63 ± 0.07	1.6 ± 0.11
Amaranth	6.44 ± 0.42	4.08 ± 0.34	2.2 ± 0.15	1.88 ± 0.17

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 47 mg/kg bw/day when Sprague Dawley female rats were treated with Amaranth dye (E123)

Executive summary:

In a repeated dose oral toxicity study, Sprague Dawley female rats were treated with Amaranth dye (E123) in the concentration of 0 and 47 mg/kg bw/day for 4 weeks.On day 14 animals were injected intraperitoneally with 1 ml/rat of 10% sheep RBCs suspension to test thedelayed type hypersensitivity response in rats.No significant effect on body weight gain was observed in treated rats as compared to control. Nosignificant effects on total leucocytes count were observed in treated rats however, a significant increase in lymphocyte and basophiles % and significant decreased in neutrophils and monocytes % were observed in treated rats as compared to control. Decrease in neutrophiles and monocytes percent could affect the integrity of the cellular immune response but not thehumoral immune response.Similarly, Increased number of circulating mononuclear cells in peripheral blood was observed but no effect on Total serum protein, albumin and globulin concentration were observed in treated rats as compared to control. In addition, Significant decreased in oedema % of the paw and increases in density of albumin band and no effect on density of globulin region were observed in treated rats as compared to control. No significant effect on relative Spleen and Thymus gland weight were observed in treated rats as compared to control. Therefore, NOAEL was considered to be47mg/kg bw/day whenSprague Dawley female rats were treated withAmaranth dye (E123) orally for 4 weeks.