Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Data is from NTRL report

Data source

Reference

Reference Type:

publication

Title:

Three Animal Safety Studies on Acid Red 1 (Red 2G)

Author:

Unilever Research U. S., IncUnilever Research U. S., Inc

Year:

1989

Bibliographic source:

Unilever Research U. S., Inc, 45 River Road, Edgewater. New Jersey 07020 OTS0516606-2, 1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Red 2G was fed to C57Bl mice to evaluate the toxic nature of the test compound in a sub-acute study conducted.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

223-098-9

EC Name:

Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

3734-67-6

Molecular formula:

C18H15N3O8S2.2Na

IUPAC Name:

disodium 5-acetamido-4-hydroxy-3-(phenyldiazenyl)naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

C.I. Acid Red 1

IUPAC Name:

C.I. Acid Red 1

Details on test material:

- Name of test material (as cited in study report): Red 2G- Molecular formula (if other than submission substance): C18H15N3O8S2.2Na- Molecular weight (if other than submission substance): 509.426 g/mol- Substance type: Organic- Physical state: No data available- Impurities (identity and concentrations): No data available

Test animals

Species:

mouse

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: No data available- Age at study initiation: No data available- Weight at study initiation: 16 to 23 g- Fasting period before study: No data available- Housing: No data available- Diet (e.g. ad libitum): No data available- Water (e.g. ad libitum): No data available- Acclimation period: No data availableENVIRONMENTAL CONDITIONS- Temperature (°C): No data available- Humidity (%):No data available- Air changes (per hr): No data available- Photoperiod (hrs dark / hrs light): No data availableIN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Red 2G, dissolved in water at appropriate concentrationsDIET 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 availableVEHICLE- Justification for use and choice of vehicle (if other than water): No data available- Concentration in vehicle: 0, 0.02%, 0.1%, 0.5% or 1.0%- 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

Details on analytical verification of doses or concentrations:

No data available

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:0, 0.02%, 0.1%, 0.5% or 1.0% (0, 20, 100, 500 or 1000 mg/Kg bw)Basis:

No. of animals per sex per dose:

Total: 50- 25 bucks and 25 does

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data- Time schedule: No data- Cage side observations checked in table [No.?] were included. No dataDETAILED CLINICAL OBSERVATIONS: No data- Time schedule: No dataBODY WEIGHT: Yes- Time schedule for examinations: No dataFOOD 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: YesFOOD 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 dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data- Time schedule for examinations: No dataOPHTHALMOSCOPIC EXAMINATION: No data - Time schedule for examinations:- Dose groups that were examined: No dataHAEMATOLOGY: Yes - Time schedule for collection of blood: On days 5, 8, 11, 17, 42 and 44- Anaesthetic used for blood collection: No data - Animals fasted: No data - How many animals: No data- Parameters checked in table [No.?] were examined. No dataCLINICAL 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 dataURINALYSIS: 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 dataNEUROBEHAVIOURAL EXAMINATION: No data- Time schedule for examinations: No data- Dose groups that were examined: No data- Battery of functions tested: No data sensory activity / grip strength / motor activity / other: No dataOTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals were subjected to full post-mortem examination immediately after death; during necropsy, liver spleen and kidneys were removed and weighed.HISTOPATHOLOGY: Yes, portions of spleen, liver, kidneys, heart, adrenals, stomach, small intestine, large intestine, bladder, lung, thymus, brain, femur, skeletal muscle and testes or uterus and ovaries were fixed in 10% formol saline. Paraffin –sections of liver, spleen and kidneys from all 50 mice were prepared by conventional methods and stained with Haematoxylin and Eosin; duplicate sections of liver, spleen and kidneys were treated by Perl's Prussian Blue Reaction (P.B.R.) to demonstrate haemosiderin. Paraffin sections of all other organs listed above were prepared from only 2 males and 2 females of each treatmont group, since experience with rats fed Red 2G suggested that no significant lesions would be detected in these organs. All organs which were not processed at this stage were retained in formol saline until histological studies had been completed and had confirmed the absence of significant lesions in organs other than spleen, liver and kidneys.During histological examination, sections of spleen, liver and kidneys were examined and scored using the “blind screening technique", whereby sections were examined at random, without reference to their group source until examination of all sections was completed. This technique was repeated three times on each section and the score finally given to each section was the average of the three recorded scores.

Other examinations:

No data available

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Clinical signs and mortality:Mortality: No death was recorded during the studyClinical signs: No data available Body weight and weight gain: No appreciable effect on body weight gain was notedFood consumption and compound intake: No appreciable effect on food intake was notedFood efficiency: No data available Water consumption and compound intake: No data available Opthalmoscopic examination: No data available Haematology: Haematological studies revealed Heinz bodies in all blood samples taken on days 5, 8, 11, 17 and 42 or 44, from mice fed 0.5 or 1.0% Red 2G. No Heinz bodies were seen at any time in blood from mice fed 0, 0.02 or 0.1% Red 2G. No significant fall in packed cell volumes was noted in male mice fed Red 2G but in females, a trend towards lower levels was recorded in animals fed dietary levels of 0.1- 1.0% After feeding the diets for six weeks, blood samples obtained from mice fed 0.5 and 1.0% Red 2G contained appreciable levels of methaemoglobin.Clinical chemistry: No data availableUrinanalysis: No data available Neurobehaviour: No data available Organ weights: An increase in splenic size in animals fed 0.5 or 1.0% Red 2G, this observation being confirmed by subsequent comparison of relative splenic weights. In these two groups, the spleens of male animals were on average, 1.5 and 2.1 times the weight of the spleens of male mice in the control group, and those of female animals 1.4 and 1.8 times the weight of the spleens of the female mice in the control group. Histological examination of the spleen was designed to determine the factor/factors associated with its increased size and weight in animals fed 0.5 or 1.0 % Red 2G.Kidney weights of mice fed Red 2G were slightly less than those of the controls.Gross pathology: All animals were in good bodily condition and no evidence of jaundice, tissue pallor or discoloration of the kidneys was observed. The most striking differences among the treatment groups were the pink of purple discoloration of the contents of the stomach, lower small intestine and urine and enlargement of the spleen. One female rat fed a diet containing 0.5% Red 2G showed a 5mm cyst, containing clear fluid, in the hilar region of the spleen.Histopathology: Spleen: Splenic enlargement and increased spleen weight was noted which was associated with increased haemosiderin deposition, increased erythropoiesis and associated sinusoidal engorgement.Well defined sex differences were observed in the levels of splenic haemosiderin, higher levels of haemosiderin being detected in female animals fed Purified Diet or Purified Diet plus Red 2G. Due to these higher background levels of haemosiderin in female mice, the detection of small additions of haemosiderin to this background pool has proved difficult, and it must be concluded that, female mice, in this trial, were a less sensitive indicator of splenic haemosiderin levels than males. This was particularly so in the case of red pulp reticular impregnation in which no significant difference was observed among the females of any treatment group.Histological examination of the splenic cyst observed in the hilar region of a female mouse, fed a diet containing 0.5% Red 2G, revealed this thin walled cyst to be lined by cuboidal/Columnar epithelium and was closely associated with a fragment of pancreas. From the location of this cyst, it seemed probable that it had originated from an occluded pancreatic duct.Liver:No evidence of toxic degenerative change or an increase in the haemosiderin content of this organ was appreciated, even in animals fed 1.0% Red 2G. No haemosiderin was observed in the cytoplasm of Kupffer or parenchymal cells of any animal in this trial while the levels of haemosiderin impregnation of sinusoidal endothelium were extremely low in all control and treatment groups. Against this very low background level of haemosiderin in control animals, any significant increase in hepatic haemosiderin could have been readily detectable and it is therefore considered that the feeding of up to 1.0% Red 2G has failed to increase the amount of this iron containing pigment in the livers of mice.Occasional tiny foci of hepatic necrosis were observed in a few mice in almost all the control and treatment groups. The incicidence and the severity of these foci were not influenced by the feeding of Red 2G.Kidneys: Cortical tubular hyaline casts, in small numbers, were noted in the majority of male mice but the number and size of these casts was not influenced by the feeding of Red 2G. In contrast, only an occasional cast was observed in female mice and these were distributed in random manner throughout the control and treatment groups.Careful scrutiny of the epithelium of the proximal convoluted tubules of the control animals revealed only small quantities of haemosiderin, this being present in finely granular form. Against this low level background, any increase in haemosiderin in this site was readily detectable.Bone Marrow:Erythroid hyperplasia of bone marrow was well defined in all mice fed 0.5 - 1.0% Red 2G but no significant lesions were observed in animals fed dietary levels of 0.02 – 0.1%.Lungs: Lymphoreticular hyperplasia, of mild to moderate severity was identified in the majority of animals and was dose independent.

Effect levels

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Average Body weight gain

Treatment group	Average body weight (g)
0.02%	22.3
0.1%	24.8
0.5%	20.3
1.0%	22.5

Table: Food consumption and compound intake

Treatment group	Average food intake in 42 days (g)	Average daily food intake (g)	Average daily Red 2G intake (mg)	Average daily Red2G intake mg/Kg bw
0.02%	592	1.41	0.28	12.5
0.1%	651	1.55	1.5	60.5
0.5%	620	1.48	1.7	354.7
1.0%	574	1.37	13.7	608.9

Table: Gross pathology

Treatment group	Stomach	Lower small intestine	Urine	Spleen
0.0%	NL	NL	NAD	NL
0.02%	Pink	Pale Pink	NAD	NL
0.1%	Bright Pink	Pink	NAD	NL
0.5%	Bright Pink	Pink	NAD	Enlarged, Dark
1.0%	Bright Pink	Purple	Pink	Enlarged, Dark

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for the test compound Red 2G is found to be 0.02% (20 mg/Kg bw).

Executive summary:

Red 2G was fed to C57Bl mice to evaluate the toxic nature of the test compound in a sub-acute study conducted.

 

Red 2G was fed to C57Bl. mice at dietary levels of 0, 0.02. 0.1. 0.5 and 1.0%(0, 20, 100, 500 or 1000 mg/Kg bw). The toxic effects included the development of Heinz bodies in red blood cells, methaemoglobinemia,. splenic enlargement, accelerated splenic erythropolesis and increased levels of haemosiderin in splenic macrophages. These findings indicated that Red 2G was capable of producing haemolysis of red blood cells in mice.

 

The No Observed Adverse Effect Level (NOAEL) for the test compound Red 2G is found to be 0.02% (20 mg/Kg bw).